Drug loaded polymeric nanoparticles and methods of making and using same

ABSTRACT

The present disclosure generally relates to methods of making nanoparticles having about 0.2 to about 35 weight percent of a therapeutic agent; and about 10 to about 99 weight percent of biocompatible polymer such as a diblock poly(lactic) acid-poly(ethylene)glycol.

RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 61/061,760, filed Jun.16, 2008; U.S. Ser. No. 61/105,916, filed Oct. 16, 2008, U.S. Ser. No.61/106,777, filed Oct. 20, 2008; U.S. Ser. No. 61/169,514, filed Apr.15, 2009; U.S. Ser. No. 61/175,209, filed May 4, 2009; U.S. Ser. No.61/061,704, filed Jun. 16, 2008; U.S. Ser. No. 61/169,519, filed Apr.15, 2009; U.S. Ser. No. 61/175,219 filed May 4, 2009; U.S. Ser. No.61/061,697, filed Jun. 16, 2008; U.S. Ser. No. 61/088,159, filed Aug.12, 2008; U.S. Ser. No. 61/169,541, filed Apr. 15, 2009; U.S. Ser. No.61/175,226, filed May 4, 2009; U.S. Ser. No. 61/173,784, filed Apr. 29,2009; U.S. Ser. No. 61/182,300, filed May 29, 2009; and U.S. Ser. No.61/173,790, filed Apr. 29, 2009; each of which is hereby incorporated byreference in their entirety.

This invention was made with United States Government support underCooperative Agreement Number 70NANB7H7021 awarded by the NationalInstitute of Standard and Technology (NIST). The United StatesGovernment has certain rights in the Invention.

BACKGROUND

Systems that deliver certain drugs to a patient (e.g., targeted to aparticular tissue or cell type or targeted to a specific diseased tissuebut not normal tissue), or that control release of drugs has long beenrecognized as beneficial.

For example, therapeutics that include an active drug and that are e.g.,targeted to a particular tissue or cell type or targeted to a specificdiseased tissue but not to normal tissue, may reduce the amount of thedrug in tissues of the body that are not targeted. This is particularlyimportant when treating a condition such as cancer where it is desirablethat a cytotoxic dose of the drug is delivered to cancer cells withoutkilling the surrounding non-cancerous tissue. Effective drug targetingmay reduce the undesirable and sometimes life threatening side effectscommon in anticancer therapy. In addition, such therapeutics may allowdrugs to reach certain tissues they would otherwise be unable to reach.

Therapeutics that offer controlled release and/or targeted therapy alsomust be able to deliver an effective amount of drug, which is a knownlimitation in other nanoparticle delivery systems. For example, it canbe a challenge to prepare nanoparticle systems that have an appropriateamount of drug associated each nanoparticle, while keeping the size ofthe nanoparticles small enough to have advantageous delivery properties.However, while it is desirable to load a nanoparticle with a highquantity of therapeutic agent, nanoparticle preparations that use a drugload that is too high will result in nanoparticles that are too largefor practical therapeutic use.

Accordingly, a need exists for nanoparticle therapeutics and methods ofmaking such nanoparticles, that are capable of delivering therapeuticlevels of drug to treat diseases such as cancer, while also reducingpatient side effects.

SUMMARY

In one aspect, the invention provides therapeutic nanoparticle thatincludes an active agent or therapeutic agent, e.g. taxane, and one,two, or three biocompatible polymers. For example, disclosed herein is atherapeutic nanoparticle comprising about 0.2 to about 35 weight percentof a therapeutic agent; about 10 to about 99 weight percent poly(lactic)acid-block-poly(ethylene)glycol copolymer or poly(lactic)-co-poly(glycolic) acid-block poly(ethylene)glycol copolymer; and about 0 toabout 50 weight percent poly(lactic) acid or poly(lactic) acid-co-poly(glycolic) acid. Exemplary therapeutic agents include antineoplasticagents such as taxanes, e.g. docetaxel and may include about 10 to about30 weight percent of a therapeutic agent, e.g., a taxane agent.

Provided herein, in part, is a method of preparing a plurality ofdisclosed therapeutic nanoparticles, comprising combining a therapeuticagent, a first polymer, and optionally a second polymer, with an organicsolvent (for example, a solvent chosen from: ethyl acetate, benzylalcohol, methylene chloride, dimethyl formamide, Tween 80 and Span 80,and combinations of two or more thereof) to form a first organic phasehaving about 5 to about 50% solids; combining the first organic phasewith a first aqueous solution (which may, in some embodiments, include areagent chosen from: sodium cholate, ethyl acetate, benzyl alcohol orcombinations thereof) to form a second phase; emulsifying the secondphase to form an emulsion phase; quenching the emulsion phase to form aquenched phase; adding a drug solubilizer to the quenched phase to forma solubilized phase of unencapsulated therapeutic agent; and filteringthe solubilized phase to recover the target-specific stealthnanoparticles, thereby forming a slurry of therapeutic nanoparticleshaving a diameter of about 80 nm to about 150 nm. In some embodiments,emulsifying the second phase may include emulsifying the second phase toform a coarse emulsion, and emulsifying the coarse emulsion to form afine emulsion phase. Emulsifying the second phase may be performed, forexample, by using a rotor stator homogenizer, probe sonicator, stir bar,or high pressure mixer. Emulsifying the coarse emulsion may be performedusing, for example, a high pressure homogenizer which may have multiple(e.g. 2, 3, 4 or more) interaction chambers, and with, for example, afeed pressure of about 2000 to about 8000, for example about 2000 toabout 6000, per interaction chamber.

In some embodiments, quenching may be at least partially performed at atemperature of about 5° C. or less, for example, at about 0° C. to about5° C. A quench:emulsion ratio may be about 8:1 to about 5:1, or about2:1 to about 40:1.

Exemplary drug solubilizers for use in the disclosed methods may includeTween 80, Tween 20, polyvinyl pyrrolidone, cyclodextran, sodium dodecylsulfate, or sodium cholate. In some embodiments, a drug solubilizer isselected from the group consisting of diethylnitrosamine, sodiumacetate, urea, glycerin, propylene glycol, glycofurol,poly(ethylene)glycol, bris(polyoxyethyleneglycolddodecyl ether, sodiumbenzoate, and sodium salicylate. The ratio of drug solubilizer totherapeutic agent may be about 100:1 to about 10:1.

In an embodiment, a method may include filtering the solubilized phasethat includes nanoparticles using e.g., tangential flow filtrationsystem. Filtering may be conducted, for example, at a first temperatureof about 0° C. to about 5° C., and then at a second temperature of about20° C. to about 30° C. Alternatively, filtering may be conducted, forexample, at a first temperature of about 20° C. to about 30° C., andthen at a second temperature of about 0° C. to about 5° C. In someembodiments, filtering comprises processing about 1 to about 6diavolumes at about 0° C. to about 5° C. and processing at least onediavolume at about 20° C. to about 30° C., for example, filtering mayinclude processing about 1 to about 6 diavolumes at about 0° C. to about5° C. and processing about one diavolume to about 15 diavolumes at about20° C. to about 30° C. In an embodiment, filtering may includeprocessing different diavolumes at different distinct temperatures. Thesolubilized phase may be purified before said filtering to substantiallyremove said organic solvent, unencapsulated therapeutic agent, and/ordrug solubilizer.

Disclosed methods may comprise sterile filtering of the slurry oftherapeutic nanoparticles using a filtration train at a controlled rate.For example, a filtration train may be used that comprises a depthfilter and a sterile filter.

Also provided herein is a method of preparing a plurality of therapeuticnanoparticles, comprising combining a therapeutic agent, a firstpolymer, and optionally a second polymer, with an organic solvent toform a first organic phase; combining the first organic phase with afirst aqueous solution to form a second phase; emulsifying the secondphase to form an emulsion phase; quenching the emulsion phase to form aquenched phase; adding a drug solubilizer to the quenched phase to forma solubilized phase of unencapsulated therapeutic agent; and filteringthe solubilized phase using tangential flow filtration with constantvolume diafiltration wherein at least one diavolume is exposed to about25° C. after a different diavolume is exposed to about −5° C. to about10° C. For example, filtering may y include processing about 2 to about5 diavolumes at about 0° C. to about 5° C., and then processing at leastone diavolume at 25° C. for at least a period of time.

Provided herein are methods of forming therapeutic nanoparticles thatmay be stable for at least 2 days at 25° C. at about 10 mg/mlconcentration. Formed therapeutic nanoparticles using disclosed methodsmay release less than 10% by weight of therapeutic agent over at least 5days at 25° C. In some embodiments, a formed therapeutic nanoparticleusing a disclosed method may e.g., encapsulate about 2 to about 30percent therapeutic agent.

In some embodiments, methods of preparing a plurality of disclosedtherapeutic nanoparticles, are provided comprising combining atherapeutic agent, a first polymer (e.g., diblock PLGA-PEG or diblockPLA-PEG copolymer), and an optional second polymer (e.g. PLAhomopolymer), and optionally a third polymer (e.g. PLA) wherein thefirst polymer or the second polymer may optionally be bound to a ligandhaving a molecular weight of less than about 1000 g/mol, for example, alow-molecular weight ligand e.g., a PSMA ligand. Such low-molecularweight PSMA ligand may be selected from the group consisting ofcompounds I, II, III and IV:

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof; wherein

m and n are each, independently, 0, 1, 2 or 3;

p is 0 or 1;

R¹, R², R⁴ and R⁵ are each, independently, selected from the groupconsisting of substituted or unsubstituted alkyl, substituted orunsubstituted aryl, and any combination thereof; and

R³ is H or CH₃;

wherein R¹, R², R⁴ or R⁵ comprise a point of covalent attachment to thenanoparticle. For example, R¹, R², R⁴ and R⁵ may be each, independently,C₁₋₆-alkyl or phenyl, or any combination of C₁₋₆-alkyl or phenyl, whichare independently substituted one or more times with OH, SH, NH₂, orCO₂H, and wherein the alkyl group may be interrupted by N(H), S or O. Inanother embodiment, for example, R¹, R², R⁴ and R⁵ are each,independently, CH₂-Ph, (CH₂)₂—SH, CH₂—SH, (CH₂)₂C(H)(NH₂)CO₂H,CH₂C(H)(NH₂)CO₂H, CH(NH₂)CH₂CO₂H, (CH₂)₂C(H)(SH)CO₂H, CH₂—N(H)-Ph,O—CH₂-Ph, or O—(CH₂)₂-Ph, wherein each Ph may be independentlysubstituted one or more times with OH, NH₂, CO₂H or SH. Exemplarylow-molecular weight PSMA ligand may be selected from the groupconsisting of

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof; and wherein the NH₂, OH or SH groups serve as thepoint of covalent attachment to the first particle, or may be selectedfrom the group consisting of

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof; wherein R is independently selected from the groupconsisting of NH₂, SH, OH, CO₂H, C₁₋₆-alkyl that is substituted withNH₂, SH, OH or CO₂H, and phenyl that is substituted with NH₂, SH, OH orCO₂H,

and wherein R serves as the point of covalent attachment to the firstpolymer. Exemplary ligands include

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof; any of which may be further substituted with NH₂, SH,OH, CO₂H, C₁₋₆-alkyl that is substituted with NH₂, SH, OH or CO₂H, orphenyl that is substituted with NH₂, SH, OH or CO₂H, wherein thesefunctional groups serve as the point of covalent attachment to the firstpolymer, for example, a low-molecular weight PSMA ligand may be

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof; wherein the NH₂ groups serve as the point of covalentattachment to the first polymer.

In some embodiments, methods of preparing a plurality of disclosedtherapeutic nanoparticles, are provided comprising combining atherapeutic agent, a first polymer (e.g., PLGA-PEG or PLA-PEG), andoptionally a second polymer (e.g. PLA, PLGA, or PEG, or copolymersthereof) an optional a third polymer (e.g. PLA or PLGA not bound to aligand). In some embodiments, the therapeutic agent is docetaxel. Inother embodiments, the therapeutic agent is selected from the groupconsisting of chemotherapeutic agents such as doxorubicin (adriamycin),mitoxantrone, gemcitabine (gemzar), daunorubicin, procarbazine,mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU),vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel(taxotere), aldesleukin, asparaginase, busulfan, carboplatin,cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethylcamptothecin (SN38),dacarbazine, S-I capecitabine, ftorafur, 5′ deoxyfluorouridine, UFT,eniluracil, deoxycytidine, 5-azacytosine, 5-azadeoxycytosine,allopurinol, 2-chloroadenosine, trimetrexate, aminopterin,methylene-10-deazaminopterin (MDAM), oxaplatin, picoplatin, tetraplatin,satraplatin, platinum-DACH, ormaplatin, CI-973, JM-216, and analogsthereof, epirubicin, etoposide phosphate, 9-aminocamptothecin,10,11-methylenedioxycamptothecin, karenitecin, 9-nitrocamptothecin, TAS103, vindesine, L-phenylalanine mustard, ifosphamidemefosphamide,perfosfamide, trophosphamide carmustine, semustine, epothilones A-E,tomudex, 6-mercaptopurine, 6-thioguanine, amsacrine, etoposidephosphate, karenitecin, acyclovir, valacyclovir, ganciclovir,amantadine, rimantadine, lamivudine, zidovudine, bevacizumab,trastuzumab, rituximab, and 5-Fluorouracil, methotrexate, budesonide,sirolimus vincristine, and combinations thereof, or the therapeuticagent may be an siRNA.

Also provided herein are methods of treating prostate cancer in asubject in need thereof, comprising administering to the subject aneffective amount of the nanoparticle prepared by the disclosed methods.

In an embodiment, also provided herein is a therapeutic nanoparticleprepared by: emulsification of a first organic phase comprising a firstpolymer and a therapeutic agent and second phase forming an emulsionphase; wherein the emulsion phase is then quenched at a temperature ofabout 0° C. to about 5° C. forming a quenched phase; and filtration ofthe quenched phase at a first temperature of about −5° C. to about 10°C.; and filtering the quenched phase at a second temperature of about25° C.; thereby forming therapeutic nanoparticles that are stable for atleast 5 days at 25° C.

A method of stabilizing therapeutic nanoparticles is also provided in anembodiment, having a therapeutic agent comprising: providing a slurrycomprising a therapeutic agent encapsulated by nanoparticles and a drugsolubilizer; filtering the slurry at a first temperature of about −5° C.to about 10° C.; filtering the slurry at a second temperature of about25° C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a pictorial representation of one embodiment of adisclosed nanoparticle.

FIG. 2 depicts an exemplary synthetic scheme to a disclosednanoparticle.

FIG. 3 is flow chart for an emulsion process for forming disclosednanoparticle.

FIG. 4 is a flow diagram for a disclosed emulsion process.

FIG. 5 depicts the effect of coarse emulsion preparation on quenchedparticle size. Placebo organic at 30% solids was used, emulsified at 5:1W:O using standard aqueous phase (1% sodium cholate, 2% benzyl alcohol,4% ethyl acetate).

FIG. 6 depicts the effect of feed pressure on resultant particle size.

FIG. 7 depicts the particle size dependence on scale.

FIG. 8 depicts the effect of solids concentration on particle size.

FIG. 9 depicts the effect of solids concentration of drug loading.

FIG. 10 depicts the effect of homopolymer PLA with PLGA-PEG or PLA-PEGon DTXL (docetaxel) loading.

FIG. 11 depicts the effect of homopolymer PLA as part of a nanoparticleon the rate of drug release of a nanoparticle.

FIG. 12 depicts the effect of cetyl alcohol on the initial rate of drugrelease of a nanoparticle.

FIG. 13 depicts in vitro release of docetaxel from disclosednanoparticles compared to conventional docetaxel

FIG. 14 depicts the effect of solids concentration and poly(lactic)homopolymer on loading percentage of sirolimus (rapamycin).

FIG. 15 depicts in vitro release of sirolimus over time for disclosednanoparticles

FIG. 16 depicts the effects of poly(lactic) homopolymer on loadingpercentage of temsirolimus.

FIG. 17 depicts the effect of solids concentration on particle size oftemsirolimus containing particles.

FIG. 18 depicts in vitro release of temsirolimus over time for disclosednanoparticles

FIG. 19 depicts in vitro release properties of an exemplary disclosednanoparticle that includes vinorelbine.

FIG. 20 depicts in vitro release properties of disclosed nanoparticlesthat include vincristine or docetaxel.

FIG. 21 depicts the pharmacokinetics of vincristine and vincristine PTNPin rats.

FIG. 22 depicts the average tumor volume after administration ofdisclosed nanoparticles that include docetaxel in a MX-1 xenograft mousemodel of breast cancer.

FIG. 23 depicts the docetaxel concentration in mouse tumors in a breastcancer MX-1 xenograft mouse model 24 hours after an intravenous dose ofdisclosed nanoparticles that include docetaxel.

FIG. 24 depicts prostate tumor distribution of disclosed nanoparticleshaving docetaxel after administration to mice inoculated with humanLNCaP prostate cancer cells.

FIG. 25 shows tumor growth suppression in mice inoculated with humanLNCaP prostate cancer cells after administration of disclosednanoparticles with docetaxel.

DETAILED DESCRIPTION

The present invention generally relates to polymeric nanoparticles thatinclude an active or therapeutic agent or drug, and methods of makingand using such therapeutic nanoparticles. In general, a “nanoparticle”refers to any particle having a diameter of less than 1000 nm, e.g.about 10 nm to about 200 nm. Disclosed therapeutic nanoparticles mayinclude nanoparticles having a diameter of about 60 to about 120 nm, orabout 70 to about 130 nm, or about 60 to about 140 nm.

Disclosed nanoparticles may include about 0.2 to about 35 weightpercent, about 3 to about 40 weight percent, about 5 to about 30 weightpercent, 10 to about 30 weight percent, 15 to 25 weight percent, or evenabout 4 to about 25 weight percent of an active agent, such asantineoplastic agent, e.g. a taxane agent (for example docetaxel).

Nanoparticles disclosed herein include one, two, three or morebiocompatible and/or biodegradable polymers. For example, a contemplatednanoparticle may include about 10 to about 99 weight percent of a one ormore block co-polymers that include a biodegradable polymer andpolyethylene glycol, and about 0 to about 50 weight percent of abiodegradable homopolymer.

In one embodiment, disclosed therapeutic nanoparticles may include atargeting ligand, e.g., a low-molecular weight PSMA ligand effective forthe treatment of a disease or disorder, such as prostate cancer, in asubject in need thereof. In certain embodiments, the low-molecularweight ligand is conjugated to a polymer, and the nanoparticle comprisesa certain ratio of ligand-conjugated polymer (e.g., PLA-PEG-Ligand) tonon-functionalized polymer (e.g. PLA-PEG or PLGA-PEG). The nanoparticlecan have an optimized ratio of these two polymers such that an effectiveamount of ligand is associated with the nanoparticle for treatment of adisease or disorder, such as cancer. For example, an increased liganddensity may increase target binding (cell binding/target uptake), makingthe nanoparticle “target specific.” Alternatively, a certainconcentration of non-functionalized polymer (e.g., non-functionalizedPLGA-PEG copolymer) in the nanoparticle can control inflammation and/orimmunogenicity (i.e., the ability to provoke an immune response), andallow the nanoparticle to have a circulation half-life that is adequatefor the treatment of a disease or disorder (e.g., prostate cancer).Furthermore, the non-functionalized polymer may, in some embodiments,lower the rate of clearance from the circulatory system via thereticuloendothelial system (RES). Thus, the non-functionalized polymermay provide the nanoparticle with characteristics that may allow theparticle to travel through the body upon administration. In someembodiments, a non-functionalized polymer may balance an otherwise highconcentration of ligands, which can otherwise accelerate clearance bythe subject, resulting in less delivery to the target cells.

For example, disclosed herein are nanoparticles that may includefunctionalized polymers conjugated to a ligand that constituteapproximately 0.1-30, e.g., 0.1-20, e.g., 0.1-10 mole percent of theentire polymer composition of the nanoparticle (i.e.,functionalized+non-functionalized polymer). Also disclosed herein, inanother embodiment, are nanoparticles that include a polymer conjugated(e.g., covalently with (i.e. through a linker (e.g. an alkylene linker)or a bond) with one or more low-molecular weight ligands, wherein theweight percent low-molecular weight ligand with respect to total polymeris between about 0.001 and 5, e.g., between about 0.001 and 2, e.g.,between about 0.001 and 1.

Also provided herein are polymeric nanoparticles that include about 2about 20 weight percent active agent. For example, a compositioncomprising such nanoparticles may be capable of delivering an effectiveamount to e.g. a target body area of a patient.

For example, disclosed nanoparticles may be able to efficiently bind toor otherwise associate with a biological entity, for example, aparticular membrane component or cell surface receptor. Targeting of atherapeutic agent (e.g., to a particular tissue or cell type, to aspecific diseased tissue but not to normal tissue, etc.) is desirablefor the treatment of tissue specific diseases such as solid tumorcancers (e.g. prostate cancer). For example, in contrast to systemicdelivery of a cytotoxic anti-cancer agent, the nanoparticles disclosedherein may substantially prevent the agent from killing healthy cells.Additionally, disclosed nanoparticles may allow for the administrationof a lower dose of the agent (as compared to an effective amount ofagent administered without disclosed nanoparticles or formulations)which may reduce the undesirable side effects commonly associated withtraditional chemotherapy.

Polymers

In some embodiments, the nanoparticles of the invention comprise amatrix of polymers and a therapeutic agent. In some embodiments, atherapeutic agent and/or targeting moiety (i.e., a low-molecular weightPSMA ligand) can be associated with at least part of the polymericmatrix. For example, in some embodiments, a targeting moiety (e.g.ligand) can be covalently associated with the surface of a polymericmatrix. In some embodiments, covalent association is mediated by alinker. The therapeutic agent can be associated with the surface of,encapsulated within, surrounded by, and/or dispersed throughout thepolymeric matrix.

A wide variety of polymers and methods for forming particles therefromare known in the art of drug delivery. In some embodiments, thedisclosure is directed toward nanoparticles with at least twomacromolecules, wherein the first macromolecule comprises a firstpolymer bound to a low-molecular weight ligand (e.g. targeting moiety);and the second macromolecule comprising a second polymer that is notbound to a targeting moiety. The nanoparticle can optionally include oneor more additional, unfunctionalized, polymers.

Any polymer can be used in accordance with the present invention.Polymers can be natural or unnatural (synthetic) polymers. Polymers canbe homopolymers or copolymers comprising two or more monomers. In termsof sequence, copolymers can be random, block, or comprise a combinationof random and block sequences. Typically, polymers in accordance withthe present invention are organic polymers.

The term “polymer,” as used herein, is given its ordinary meaning asused in the art, i.e., a molecular structure comprising one or morerepeat units (monomers), connected by covalent bonds. The repeat unitsmay all be identical, or in some cases, there may be more than one typeof repeat unit present within the polymer. In some cases, the polymercan be biologically derived, i.e., a biopolymer. Non-limiting examplesinclude peptides or proteins. In some cases, additional moieties mayalso be present in the polymer, for example biological moieties such asthose described below. If more than one type of repeat unit is presentwithin the polymer, then the polymer is said to be a “copolymer.” It isto be understood that in any embodiment employing a polymer, the polymerbeing employed may be a copolymer in some cases. The repeat unitsforming the copolymer may be arranged in any fashion. For example, therepeat units may be arranged in a random order, in an alternating order,or as a block copolymer, i.e., comprising one or more regions eachcomprising a first repeat unit (e.g., a first block), and one or moreregions each comprising a second repeat unit (e.g., a second block),etc. Block copolymers may have two (a diblock copolymer), three (atriblock copolymer), or more numbers of distinct blocks.

Disclosed particles can include copolymers, which, in some embodiments,describes two or more polymers (such as those described herein) thathave been associated with each other, usually by covalent bonding of thetwo or more polymers together. Thus, a copolymer may comprise a firstpolymer and a second polymer, which have been conjugated together toform a block copolymer where the first polymer can be a first block ofthe block copolymer and the second polymer can be a second block of theblock copolymer. Of course, those of ordinary skill in the art willunderstand that a block copolymer may, in some cases, contain multipleblocks of polymer, and that a “block copolymer,” as used herein, is notlimited to only block copolymers having only a single first block and asingle second block. For instance, a block copolymer may comprise afirst block comprising a first polymer, a second block comprising asecond polymer, and a third block comprising a third polymer or thefirst polymer, etc. In some cases, block copolymers can contain anynumber of first blocks of a first polymer and second blocks of a secondpolymer (and in certain cases, third blocks, fourth blocks, etc.). Inaddition, it should be noted that block copolymers can also be formed,in some instances, from other block copolymers. For example, a firstblock copolymer may be conjugated to another polymer (which may be ahomopolymer, a biopolymer, another block copolymer, etc.), to form a newblock copolymer containing multiple types of blocks, and/or to othermoieties (e.g., to non-polymeric moieties).

In some embodiments, the polymer (e.g., copolymer, e.g., blockcopolymer) can be amphiphilic, i.e., having a hydrophilic portion and ahydrophobic portion, or a relatively hydrophilic portion and arelatively hydrophobic portion. A hydrophilic polymer can be onegenerally that attracts water and a hydrophobic polymer can be one thatgenerally repels water. A hydrophilic or a hydrophobic polymer can beidentified, for example, by preparing a sample of the polymer andmeasuring its contact angle with water (typically, the polymer will havea contact angle of less than 60°, while a hydrophobic polymer will havea contact angle of greater than about) 60°. In some cases, thehydrophilicity of two or more polymers may be measured relative to eachother, i.e., a first polymer may be more hydrophilic than a secondpolymer. For instance, the first polymer may have a smaller contactangle than the second polymer.

In one set of embodiments, a polymer (e.g., copolymer, e.g., blockcopolymer) contemplated herein includes a biocompatible polymer, i.e.,the polymer that does not typically induce an adverse response wheninserted or injected into a living subject, for example, withoutsignificant inflammation and/or acute rejection of the polymer by theimmune system, for instance, via a T-cell response. Accordingly, thetherapeutic particles contemplated herein can be non-immunogenic. Theterm non-immunogenic as used herein refers to endogenous growth factorin its native state which normally elicits no, or only minimal levelsof, circulating antibodies, T-cells, or reactive immune cells, and whichnormally does not elicit in the individual an immune response againstitself.

Biocompatibility typically refers to the acute rejection of material byat least a portion of the immune system, i.e., a nonbiocompatiblematerial implanted into a subject provokes an immune response in thesubject that can be severe enough such that the rejection of thematerial by the immune system cannot be adequately controlled, and oftenis of a degree such that the material must be removed from the subject.One simple test to determine biocompatibility can be to expose a polymerto cells in vitro; biocompatible polymers are polymers that typicallywill not result in significant cell death at moderate concentrations,e.g., at concentrations of 50 micrograms/10⁶ cells. For instance, abiocompatible polymer may cause less than about 20% cell death whenexposed to cells such as fibroblasts or epithelial cells, even ifphagocytosed or otherwise uptaken by such cells. Non-limiting examplesof biocompatible polymers that may be useful in various embodiments ofthe present invention include polydioxanone (PDO), polyhydroxyalkanoate,polyhydroxybutyrate, poly(glycerol sebacate), polyglycolide,polylactide, PLGA, polycaprolactone, or copolymers or derivativesincluding these and/or other polymers.

In certain embodiments, contemplated biocompatible polymers may bebiodegradable, i.e., the polymer is able to degrade, chemically and/orbiologically, within a physiological environment, such as within thebody. As used herein, “biodegradable” polymers are those that, whenintroduced into cells, are broken down by the cellular machinery(biologically degradable) and/or by a chemical process, such ashydrolysis, (chemically degradable) into components that the cells caneither reuse or dispose of without significant toxic effect on thecells. In one embodiment, the biodegradable polymer and theirdegradation byproducts can be biocompatible.

For instance, a contemplated polymer may be one that hydrolyzesspontaneously upon exposure to water (e.g., within a subject), thepolymer may degrade upon exposure to heat (e.g., at temperatures ofabout 37° C.). Degradation of a polymer may occur at varying rates,depending on the polymer or copolymer used. For example, the half-lifeof the polymer (the time at which 50% of the polymer can be degradedinto monomers and/or other nonpolymeric moieties) may be on the order ofdays, weeks, months, or years, depending on the polymer. The polymersmay be biologically degraded, e.g., by enzymatic activity or cellularmachinery, in some cases, for example, through exposure to a lysozyme(e.g., having relatively low pH). In some cases, the polymers may bebroken down into monomers and/or other nonpolymeric moieties that cellscan either reuse or dispose of without significant toxic effect on thecells (for example, polylactide may be hydrolyzed to form lactic acid,polyglycolide may be hydrolyzed to form glycolic acid, etc.).

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEGylated polymers andcopolymers of lactide and glycolide (e.g., PEGylated PLA, PEGylated PGA,PEGylated PLGA, and derivatives thereof. In some embodiments, polyestersinclude, for example, polyanhydrides, poly(ortho ester) PEGylatedpoly(ortho ester), poly(caprolactone), PEGylated poly(caprolactone),polylysine, PEGylated polylysine, poly(ethylene imine), PEGylatedpoly(ethylene imine), poly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester), poly[α-(4-aminobutyl)-L-glycolic acid],and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA can be characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid-glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention can be characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85. In some embodiments, theratio of lactic acid to glycolic acid monomers in the polymer of theparticle (e.g., the PLGA block copolymer or PLGA-PEG block copolymer),may be selected to optimize for various parameters such as water uptake,therapeutic agent release and/or polymer degradation kinetics can beoptimized.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid polyacrylamide, amino alkyl methacrylatecopolymer, glycidyl methacrylate copolymers, polycyanoacrylates, andcombinations comprising one or more of the foregoing polymers. Theacrylic polymer may comprise fully-polymerized copolymers of acrylic andmethacrylic acid esters with a low content of quaternary ammoniumgroups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g. DNA, RNA, or derivatives thereof).Amine-containing polymers such as poly(lysine), polyethylene imine(PEI), and poly(amidoamine) dendrimers are contemplated for use, in someembodiments, in a disclosed particle.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains. Examples of these polyesters includepoly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester).

Particles disclosed herein may or may not contain PEG. In addition,certain embodiments can be directed towards copolymers containingpoly(ester-ether)s, e.g., polymers having repeat units joined by esterbonds (e.g., R—C(O)—O—R′ bonds) and ether bonds (e.g., R—O—R′ bonds). Insome embodiments of the invention, a biodegradable polymer, such as ahydrolyzable polymer, containing carboxylic acid groups, may beconjugated with poly(ethylene glycol) repeat units to form apoly(ester-ether). A polymer (e.g., copolymer, e.g., block copolymer)containing poly(ethylene glycol) repeat units can also be referred to asa “PEGylated” polymer.

It is contemplated that PEG may include a terminal end group, forexample, when PEG is not conjugated to a ligand. For example, PEG mayterminate in a hydroxyl, a methoxy or other alkoxyl group, a methyl orother alkyl group, an aryl group, a carboxylic acid, an amine, an amide,an acetyl group, a guanidino group, or an imidazole. Other contemplatedend groups include azide, alkyne, maleimide, aldehyde, hydrazide,hydroxylamine, alkoxyamine, or thiol moieties.

Those of ordinary skill in the art will know of methods and techniquesfor PEGylating a polymer, for example, by using EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS(N-hydroxysuccinimide) to react a polymer to a PEG group terminating inan amine, by ring opening polymerization techniques (ROMP), or the like.

In one embodiment, the molecular weight of the polymers can be optimizedfor effective treatment as disclosed herein. For example, the molecularweight of a polymer may influence particle degradation rate (such aswhen the molecular weight of a biodegradable polymer can be adjusted),solubility, water uptake, and drug release kinetics. For example, themolecular weight of the polymer can be adjusted such that the particlebiodegrades in the subject being treated within a reasonable period oftime (ranging from a few hours to 1-2 weeks, 3-4 weeks, 5-6 weeks, 7-8weeks, etc.). A disclosed particle can for example comprise a diblockcopolymer of PEG and PL(G)A, wherein for example, the PEG portion mayhave a number average molecular weight of about 1,000-20,000, e.g.,about 2,000-20,000, e.g., about 2 to about 10,000, and the PL(G)Aportion may have a number average molecular weight of about 5,000 toabout 20,000, or about 5,000-100,000, e.g., about 20,000-70,000, e.g.,about 15,000-50,000.

For example, disclosed here is an exemplary therapeutic nanoparticlethat includes about 10 to about 99 weight percent poly(lactic)acid-block-poly(ethylene)glycol copolymer or poly(lactic)-co-poly(glycolic) acid-block-poly(ethylene)glycol copolymer, or about 20 toabout 80 weight percent, about 40 to about 80 weight percent, or about30 to about 50 weight percent, or about 70 to about 90 weight percentpoly(lactic) acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly(glycolic) acid-poly(ethylene)glycol copolymer. Exemplary poly(lactic)acid-poly(ethylene)glycol copolymers can include a number averagemolecular weight of about 15 to about 20 kDa, or about 10 to about 25kDa of poly(lactic) acid and a number average molecular weight of about4 to about 6, or about 2 kDa to about 10 kDa of poly(ethylene)glycol.

Disclosed nanoparticles may optionally include about 1 to about 50weight percent poly(lactic) acid or poly(lactic) acid-co-poly (glycolic)acid (which does not include PEG), or may optionally include about 1 toabout 50 weight percent, or about 10 to about 50 weight percent or about30 to about 50 weight percent poly(lactic) acid or poly(lactic)acid-co-poly (glycolic) acid. For example, poly(lactic) orpoly(lactic)-co-poly(glycolic) acid may have a number average moleculeweight of about 5 to about 15 kDa, or about 5 to about 12 kDa. ExemplaryPLA may have a number average molecular weight of about 5 to about 10kDa. Exemplary PLGA may have a number average molecular weight of about8 to about 12 kDa.

In certain embodiments, the polymers of the nanoparticles can beconjugated to a lipid. The polymer can be, for example, alipid-terminated PEG. As described below, the lipid portion of thepolymer can be used for self assembly with another polymer, facilitatingthe formation of a nanoparticle. For example, a hydrophilic polymercould be conjugated to a lipid that will self assemble with ahydrophobic polymer.

In some embodiments, lipids are oils. In general, any oil known in theart can be conjugated to the polymers used in the invention. In someembodiments, an oil can comprise one or more fatty acid groups or saltsthereof. In some embodiments, a fatty acid group can comprisedigestible, long chain (e.g., C₈-C₅₀), substituted or unsubstitutedhydrocarbons. In some embodiments, a fatty acid group can be a C₁₀-C₂₀fatty acid or salt thereof. In some embodiments, a fatty acid group canbe a C₁₅-C₂₀ fatty acid or salt thereof. In some embodiments, a fattyacid can be unsaturated. In some embodiments, a fatty acid group can bemonounsaturated. In some embodiments, a fatty acid group can bepolyunsaturated. In some embodiments, a double bond of an unsaturatedfatty acid group can be in the cis conformation. In some embodiments, adouble bond of an unsaturated fatty acid can be in the transconformation.

In some embodiments, a fatty acid group can be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group can be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In a particular embodiment, the lipid is of the Formula V:

and salts thereof, wherein each R is, independently, C₁₋₃₀ alkyl. In oneembodiment of Formula V, the lipid is 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof,e.g., the sodium salt.

In one embodiment, optional small molecule targeting moieties arebonded, e.g., covalently bonded, to the lipid component of thenanoparticle. For example, provided herein is a nanoparticle comprisinga therapeutic agent, a polymeric matrix comprising functionalized andnon-functionalized polymers, and lipid, and a low-molecular weight PSMAtargeting ligand, wherein the targeting ligand is bonded, e.g.,covalently bonded, to the lipid component of the nanoparticle. In oneembodiment, the lipid component that is bonded to the low-molecularweight targeting moiety is of the Formula V. In another embodiment, theinvention provides a target-specific nanoparticle comprising atherapeutic agent, a polymeric matrix, DSPE, and a low-molecular weightPSMA targeting ligand, wherein the ligand is bonded, e.g., covalentlybonded, to DSPE. For example, the nanoparticle of the invention maycomprise a polymeric matrix comprising PLGA-DSPE-PEG-Ligand.

A contemplated nanoparticle may include a ratio of ligand-bound polymerto non-functionalized polymer effective for the treatment of prostatecancer, wherein the hydrophilic, ligand-bound polymer is conjugated to alipid that will self assemble with the hydrophobic polymer, such thatthe hydrophobic and hydrophilic polymers that constitute thenanoparticle are not covalently bound. “Self-assembly” refers to aprocess of spontaneous assembly of a higher order structure that relieson the natural attraction of the components of the higher orderstructure (e.g., molecules) for each other. It typically occurs throughrandom movements of the molecules and formation of bonds based on size,shape, composition, or chemical properties. For example, such a methodcomprises providing a first polymer that is reacted with a lipid, toform a polymer/lipid conjugate. The polymer/lipid conjugate is thenreacted with the low-molecular weight ligand to prepare a ligand-boundpolymer/lipid conjugate; and mixing the ligand-bound polymer/lipidconjugate with a second, non-functionalized polymer, and the therapeuticagent; such that the nanoparticle is formed. In certain embodiments, thefirst polymer is PEG, such that a lipid-terminated PEG is formed. In oneembodiment, the lipid is of the Formula V, e.g., 2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof,e.g., the sodium salt. The lipid-terminated PEG can then, for example,be mixed with PLGA to form a nanoparticle.

Targeting Moieties

Provided herein are nanoparticles that may include an optional targetingmoiety, i.e., a moiety able to bind to or otherwise associate with abiological entity, for example, a membrane component, a cell surfacereceptor, prostate specific membrane antigen, or the like. A targetingmoiety present on the surface of the particle may allow the particle tobecome localized at a particular targeting site, for instance, a tumor,a disease site, a tissue, an organ, a type of cell, etc. As such, thenanoparticle may then be “target specific.” The drug or other payloadmay then, in some cases, be released from the particle and allowed tointeract locally with the particular targeting site.

In one embodiment, a disclosed nanoparticle includes a targeting moietythat is a low-molecular weight ligand, e.g., a low-molecular weight PSMAligand. The term “bind” or “binding,” as used herein, refers to theinteraction between a corresponding pair of molecules or portionsthereof that exhibit mutual affinity or binding capacity, typically dueto specific or non-specific binding or interaction, including, but notlimited to, biochemical, physiological, and/or chemical interactions.“Biological binding” defines a type of interaction that occurs betweenpairs of molecules including proteins, nucleic acids, glycoproteins,carbohydrates, hormones, or the like. The term “binding partner” refersto a molecule that can undergo binding with a particular molecule.“Specific binding” refers to molecules, such as polynucleotides, thatare able to bind to or recognize a binding partner (or a limited numberof binding partners) to a substantially higher degree than to other,similar biological entities. In one set of embodiments, the targetingmoiety has an affinity (as measured via a disassociation constant) ofless than about 1 micromolar, at least about 10 micromolar, or at leastabout 100 micromolar.

For example, a targeting portion may cause the particles to becomelocalized to a tumor (e.g. a solid tumor) a disease site, a tissue, anorgan, a type of cell, etc. within the body of a subject, depending onthe targeting moiety used. For example, a low-molecular weight PSMAligand may become localized to a solid tumor, e.g. breast or prostatetumors or cancer cells. The subject may be a human or non-human animal.Examples of subjects include, but are not limited to, a mammal such as adog, a cat, a horse, a donkey, a rabbit, a cow, a pig, a sheep, a goat,a rat, a mouse, a guinea pig, a hamster, a primate, a human or the like.

Contemplated targeting moieties include small molecules. In certainembodiments, the term “small molecule” refers to organic compounds,whether naturally-occurring or artificially created (e.g., via chemicalsynthesis) that have relatively low molecular weight and that are notproteins, polypeptides, or nucleic acids. Small molecules typically havemultiple carbon-carbon bonds. In certain embodiments, small moleculesare less than about 2000 g/mol in size. In some embodiments, smallmolecules are less than about 1500 g/mol or less than about 1000 g/mol.In some embodiments, small molecules are less than about 800 g/mol orless than about 500 g/mol, for example about 100 g/mol to about 600g/mol, or about 200 g/mol to about 500 g/mol.

For example, a targeting moiety may small target prostate cancer tumors,for example a target moiety may be PSMA peptidase inhibitor. Thesemoieties are also referred to herein as “low-molecular weight PSMAligands.” When compared with expression in normal tissues, expression ofprostate specific membrane antigen (PSMA) is at least 10-foldoverexpressed in malignant prostate relative to normal tissue, and thelevel of PSMA expression is further up-regulated as the diseaseprogresses into metastatic phases (Silver et al. 1997, Clin. CancerRes., 3:81).

In some embodiments, the low-molecular weight PSMA ligand is of theFormulae I, II, III or IV:

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof;

wherein m and n are each, independently, 0, 1, 2 or 3; p is 0 or 1;

R¹, R², R⁴ and R⁵ are each, independently, selected from the groupconsisting of substituted or unsubstituted alkyl (e.g., C₁₋₁₀-alkyl,C₁₋₆-alkyl, or C₁₋₄-alkyl), substituted or unsubstituted aryl (e.g.,phenyl or pyrdinyl), and any combination thereof; and R³ is H orC₁₋₆-alkyl (e.g., CH₃).

For compounds of Formulae I, II, III and IV, R¹, R², R⁴ or R⁵ comprisepoints of attachment to the nanoparticle, e.g., a point of attachment toa polymer that forms part of a disclosed nanoparticle, e.g., PEG. Thepoint of attachment may be formed by a covalent bond, ionic bond,hydrogen bond, a bond formed by adsorption including chemical adsorptionand physical adsorption, a bond formed from van der Waals bonds, ordispersion forces. For example, if R¹, R², R⁴ or R⁵ are defined as ananiline or C₁₋₆-alkyl-NH₂ group, any hydrogen (e.g., an amino hydrogen)of these functional groups could be removed such that the low-molecularweight PSMA ligand is covalently bound to the polymeric matrix (e.g.,the PEG-block of the polymeric matrix) of the nanoparticle. As usedherein, the term “covalent bond” refers to a bond between two atomsformed by sharing at least one pair of electrons.

In particular embodiments of the Formulae I, II, III or IV, R¹, R², R⁴and R⁵ are each, independently, C₁₋₆-alkyl or phenyl, or any combinationof C₁₋₆-alkyl or phenyl, which are independently substituted one or moretimes with OH, SH, NH₂, or CO₂H, and wherein the alkyl group may beinterrupted by N(H), S or O. In another embodiment, R¹, R², R⁴ and R⁵are each, independently, CH₂-Ph, (CH₂)₂—SH, CH₂—SH, (CH₂)₂C(H)(NH₂)CO₂H,CH₂C(H)(NH₂)CO₂H, CH(NH₂)CH₂CO₂H, (CH₂)₂C(H)(SH)CO₂H, CH₂—N(H)-Ph,O—CH₂-Ph, or O—(CH₂)₂-Ph, wherein each Ph may be independentlysubstituted one or more times with OH, NH₂, CO₂H or SH. For theseformulae, the NH₂, OH or SH groups serve as the point of covalentattachment to the nanoparticle (e.g., —N(H)-PEG, —O-PEG, or —S-PEG).

In still another embodiment, the low-molecular weight PSMA ligand isselected from the group consisting of

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof, and wherein the NH₂, OH or SH groups serve as thepoint of covalent attachment to the nanoparticle (e.g., —N(H)-PEG,—O-PEG, or —S-PEG).

In another embodiment, the low-molecular weight PSMA ligand is selectedfrom the group consisting of

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof, wherein R is independently selected from the groupconsisting of NH₂, SH, OH, CO₂H, C₁₋₆-alkyl that is substituted withNH₂, SH, OH or CO₂H, and phenyl that is substituted with NH₂, SH, OH orCO₂H, and wherein R serves as the point of covalent attachment to thenanoparticle (e.g., —N(H)-PEG, —S-PEG, —O-PEG, or CO₂-PEG).

In another embodiment, the low-molecular weight PSMA ligand is selectedfrom the group consisting of

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof, wherein the NH₂ or CO₂H groups serve as the point ofcovalent attachment to the nanoparticle (e.g., —N(H)-PEG, or CO₂-PEG).These compounds may be further substituted with NH₂, SH, OH, CO₂H,C₁₋₆-alkyl that is substituted with NH₂, SH, OH or CO₂H, or phenyl thatis substituted with NH₂, SH, OH or CO₂H, wherein these functional groupscan also serve as the point of covalent attachment to the nanoparticle.

In another embodiment, the low-molecular weight PSMA ligand is

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof, wherein n is 1, 2, 3, 4, 5 or 6. For this ligand, theNH₂ group serves as the point of covalent attachment to the nanoparticle(e.g., —N(H)-PEG).

In still another embodiment, the low-molecular weight PSMA ligand is

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof. Particularly, the butyl-amine compound has theadvantage of ease of synthesis, especially because of its lack of abenzene ring. Furthermore, without wishing to be bound by theory, thebutyl-amine compound will likely break down into naturally occurringmolecules (i.e., lysine and glutamic acid), thereby minimizing toxicityconcerns.

In some embodiments, small molecule targeting moieties that may be usedto target cells associated with solid tumors such as prostate or breastcancer tumors include PSMA peptidase inhibitors such as 2-PMPA, GPI5232,VA-033, phenylalkylphosphonamidates and/or analogs and derivativesthereof. In some embodiments, small molecule targeting moieties that maybe used to target cells associated with prostate cancer tumors includethiol and indole thiol derivatives, such as 2-MPPA and3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid derivatives. In someembodiments, small molecule targeting moieties that may be used totarget cells associated with prostate cancer tumors include hydroxamatederivatives. In some embodiments, small molecule targeting moieties thatmay be used to target cells associated with prostate cancer tumorsinclude PBDA- and urea-based inhibitors, such as ZJ 43, ZJ 11, ZJ 17, ZJ38 and/or and analogs and derivatives thereof, androgen receptortargeting agents (ARTAs), polyamines, such as putrescine, spermine, andspermidine, inhibitors of the enzyme glutamate carboxylase II (GCPII),also known as NAAG Peptidase or NAALADase.

In another embodiment of the instant invention, the targeting moiety canbe a ligand that targets Her2, EGFR, or toll receptors.

For example, contemplated the targeting moieties may include a nucleicacid, polypeptide, glycoprotein, carbohydrate, or lipid. For example, atargeting moiety can be a nucleic acid targeting moiety (e.g. anaptamer, e.g., the A10 aptamer) that binds to a cell type specificmarker. In general, an aptamer is an oligonucleotide (e.g., DNA, RNA, oran analog or derivative thereof) that binds to a particular target, suchas a polypeptide. In some embodiments, a targeting moiety may be anaturally occurring or synthetic ligand for a cell surface receptor,e.g., a growth factor, hormone, LDL, transferrin, etc. A targetingmoiety can be an antibody, which term is intended to include antibodyfragments, characteristic portions of antibodies, single chain targetingmoieties can be identified, e.g., using procedures such as phagedisplay.

Targeting moieties may be a targeting peptide or targetingpeptidomimetic has a length of up to about 50 residues. For example, atargeting moieties may include the amino acid sequence AKERC, CREKA,ARYLQKLN or AXYLZZLN, wherein X and Z are variable amino acids, orconservative variants or peptidomimetics thereof. In particularembodiments, the targeting moiety is a peptide that includes the aminoacid sequence AKERC, CREKA, ARYLQKLN or AXYLZZLN, wherein X and Z arevariable amino acids, and has a length of less than 20, 50 or 100residues. The CREKA (Cys Arg Glu Lys Ala) peptide or a peptidomimeticthereof peptide or the octapeptide AXYLZZLN are also comtemplated astargeting moities, as well as peptides, or conservative variants orpeptidomimetics thereof, that binds or forms a complex with collagen IV,or the targets tissue basement membrane (e.g., the basement membrane ofa blood vessel), can be used as a targeting moiety. Exemplary targetingmoieties include peptides that target ICAM (intercellular adhesionmolecule, e.g. ICAM-1).

Targeting moieties disclosed herein are typically conjugated to adisclosed polymer or copolymer (e.g. PLA-PEG), and such a polymerconjugate may form part of a disclosed nanoparticle. For example, adisclosed therapeutic nanoparticle may optionally include about 0.2 toabout 10 weight percent of a PLA-PEG or PLGA-PEG, wherein the PEG isfunctionalized with a targeting ligand (e.g. PLA-PEG-Ligand).Contemplated therapeutic nanoparticles may include, for example, about0.2 to about 10 mole percent PLA-PEG-GL2 or poly (lactic) acid -co poly(glycolic) acid-PEG-GL2. For example, PLA-PEG-GL2 may include a numberaverage molecular weight of about 10 kDa to about 20 kDa and a numberaverage molecular weight of about 4,000 to about 8,000.

Such a targeting ligand may be, in some embodiments, covalently bound tothe PEG, for example, bound to the PEG via an alkylene linker, e.g.PLA-PEG-alkylene-GL2. For example, a disclosed nanoparticle may includeabout 0.2 to about 10 mole percent PLA-PEG-GL2 or poly (lactic) acid copoly (glycolic) acid-PEG-GL2. It is understood that reference toPLA-PEG-GL2 or PLGA-PEG-GL2 refers to moieties that may include analkylene linker (e.g. C₁-C₂₀, e.g., (CH₂)₅) linking a PLA-PEG orPLGA-PEG to GL2.

Exemplary polymeric conjugates include:

wherein R₁ is selected from the group consisting of H, and a C₁-C₂₀alkyl group optionally substituted with one, two, three or morehalogens;

R₂ is a bond, an ester linkage, or amide linkage;

R₃ is an C₁-C₁₀ alkylene or a bond;

x is 50 to about 1500, or about 60 to about 1000;

y is 0 to about 50; and

z is about 30 to about 200, or about 50 to about 180.

In a different embodiment, x represents 0 to about 1 mole fraction; andy may represent about 0 to about 0.5 mole fraction. In an exemplaryembodiment, x+y may be about 20 to about 1720, and/or z may be about 25to about 455.

For example, a disclosed nanoparticle may include a polymeric targetingmoiety represented by Formula VI:

wherein n is about 200 to about 300, e.g., about 222, and m is about 80to about 130, e.g. about 114. Disclosed nanoparticles, in certainembodiments, may include about 0.1 to about 4% by weight of e.g. apolymeric conjugate of formula VI, or about 0.1 to about 2% or about 0.1to about 1%, or about 0.2% to about 0.8% by weight of e.g., a polymericconjugate of formula VI.

In an exemplary embodiment, a disclosed nanoparticle comprises ananoparticle having a PLA-PEG-alkylene-GL2 conjugate, where, forexample, PLA has a number average molecular weight of about 16,000 Da,PEG has a molecular weight of about 5000 Da, and e.g., the alkylenelinker is a C₁-C₂₀ alkylene, e.g. (CH₂)₅.

For example, a disclosed nanoparticle may include a conjugaterepresented by:

where y is about 222 and z is about 114.

A disclosed polymeric conjugate may be formed using any suitableconjugation technique. For instance, two compounds such as a targetingmoiety and a biocompatible polymer, a biocompatible polymer and apoly(ethylene glycol), etc., may be conjugated together using techniquessuch as EDC-NHS chemistry (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide) or a reactioninvolving a maleimide or a carboxylic acid, which can be conjugated toone end of a thiol, an amine, or a similarly functionalized polyether.The conjugation of such polymers, for instance, the conjugation of apoly(ester) and a poly(ether) to form a poly(ester-ether), can beperformed in an organic solvent, such as, but not limited to,dichloromethane, acetonitrile, chloroform, dimethylformamide,tetrahydrofuran, acetone, or the like. Specific reaction conditions canbe determined by those of ordinary skill in the art using no more thanroutine experimentation.

In another set of embodiments, a conjugation reaction may be performedby reacting a polymer that comprises a carboxylic acid functional group(e.g., a poly(ester-ether) compound) with a polymer or other moiety(such as a targeting moiety) comprising an amine. For instance, atargeting moiety, such as a low-molecular weight PSMA ligand, may bereacted with an amine to form an amine-containing moiety, which can thenbe conjugated to the carboxylic acid of the polymer. Such a reaction mayoccur as a single-step reaction, i.e., the conjugation is performedwithout using intermediates such as N-hydroxysuccinimide or a maleimide.The conjugation reaction between the amine-containing moiety and thecarboxylic acid-terminated polymer (such as a poly(ester-ether)compound) may be achieved, in one set of embodiments, by adding theamine-containing moiety, solubilized in an organic solvent such as (butnot limited to) dichloromethane, acetonitrile, chloroform,tetrahydrofuran, acetone, formamide, dimethylformamide, pyridines,dioxane, or dimethysulfoxide, to a solution containing the carboxylicacid-terminated polymer. The carboxylic acid-terminated polymer may becontained within an organic solvent such as, but not limited to,dichloromethane, acetonitrile, chloroform, dimethylformamide,tetrahydrofuran, or acetone. Reaction between the amine-containingmoiety and the carboxylic acid-terminated polymer may occurspontaneously, in some cases. Unconjugated reactants may be washed awayafter such reactions, and the polymer may be precipitated in solventssuch as, for instance, ethyl ether, hexane, methanol, or ethanol.

As a specific example, a low-molecular weight PSMA ligand may beprepared as a targeting moiety in a particle as follows. Carboxylic acidmodified poly(lactide-co-glycolide) (PLGA-COOH) may be conjugated to anamine-modified heterobifunctional poly(ethylene glycol) (NH₂-PEG-COOH)to form a copolymer of PLGA-PEG-COOH. By using an amine-modifiedlow-molecular weight PSMA ligand (NH₂-Lig), a triblock polymer ofPLGA-PEG-Lig may be formed by conjugating the carboxylic acid end of thePEG to the amine functional group on the ligand. The multiblock polymercan then be used, for instance, as discussed below, e.g., fortherapeutic applications.

As used herein, the term “alkyl” includes saturated aliphatic groups,including straight-chain alkyl groups (e.g., methyl, ethyl, propyl,butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chainalkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl(alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups.

The term “aryl” includes groups, including 5- and 6-membered single-ringaromatic groups that can include from zero to four heteroatoms, forexample, phenyl, pyrrole, furan, thiophene, thiazole, isothiaozole,imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine,pyrazine, pyridazine, and pyrimidine, and the like. Furthermore, theterm “aryl” includes multicyclic aryl groups, e.g., tricyclic, bicyclic,e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole,benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline,isoquinoline, anthryl, phenanthryl, napthridine, indole, benzofuran,purine, benzofuran, deazapurine, or indolizine. Those aryl groups havingheteroatoms in the ring structure can also be referred to as “arylheterocycles”, “heterocycles,” “heteroaryls” or “heteroaromatics.” Thearomatic ring can be substituted at one or more ring positions with suchsubstituents as described above, as for example, alkyl, halogen,hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminoacarbonyl,aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino(including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Arylgroups can also be fused or bridged with alicyclic or heterocyclic ringswhich are not aromatic so as to form a polycycle (e.g., tetralin).

Targeting moities can be, for example, further substituted with afunctional group that can be reacted with a polymer of the invention(e.g., PEG) in order to produce a polymer conjugated to a targetingmoiety. The functional groups include any moiety that can be used tocreate a covalent bond with a polymer (e.g., PEG), such as amino,hydroxy, and thio. In a particular embodiment, the small molecules canbe substituted with NH₂, SH or OH, which are either bound directly tothe small molecule, or bound to the small molecule via an additionalgroup, e.g., alkyl or phenyl. In a non-limiting example, the smallmolecules disclosed in the patents, patent applications, and non-patentreferences cited herein may be bound to aniline, alkyl-NH₂ (e.g.,(CH₂)₁₋₆NH₂), or alkyl-SH (e.g., (CH₂)₁₋₆NH₂), wherein the NH₂ and SHgroups may be reacted with a polymer (e.g., PEG), to form a covalentbond with that polymer, i.e., to form a polymeric conjugate.

For example, disclosed herein is a nanoparticle having a therapeuticagent; and a first macromolecule comprising a PLGA-PEG copolymer orPLA-PEG copolymer that is conjugated to ligand having a molecular weightbetween about 100 g/mol and 500 g/mol wherein the PLGA-PEG copolymer orPLA-PEG copolymer that is conjugated to ligand is about 0.1 to about 30mole percent of the total polymer content, or about 0.1 to about 20 molepercent, or about 0.1 to about 10 mole percent, or about 1 to about 5mole percent of the total polymer content of a nanoparticle. Such ananoparticle may further include a second macromolecule comprising aPLGA-PEG copolymer or PLA-PEG copolymer, wherein the copolymer is notbound to a targeting moiety; and a pharmaceutically acceptableexcipient. For example, the first copolymer may have about 0.001 and 5weight percent of the ligand with respect to total polymer content.

Exemplary nanoparticles may include a therapeutic agent; and a polymercomposition, wherein the polymer composition comprises: a firstmacromolecule comprising first polymer bound to a ligand; and a secondmacromolecule comprising a second polymer not bound to a targetingmoiety; wherein the polymer composition comprises about 0.001 to about5.0 weight percent of said ligand. Such ligands may have a molecularweight of about 100 g/mol to about 6000 g/mol, or less than about 1000g/mol, e.g. about 100 g/mole to about 500 g/mol. In another embodiment,provided herein is a pharmaceutical composition, comprising a pluralityof target-specific polymeric nanoparticles each comprising a therapeuticagent; and a polymer composition, wherein the polymer compositioncomprises about 0.1 to about 30 mole percent, or about 0.1 to about 20mole percent, or about 0.1 to about 10 mole percent of a firstmacromolecule comprising first polymer bound to a ligand; and a secondmacromolecule comprising a second polymer not bound to a targetingmoiety; and a pharmaceutically acceptable excipient.

Nanoparticles

Disclosed nanoparticles may have a substantially spherical (i.e., theparticles generally appear to be spherical), or non-sphericalconfiguration. For instance, the particles, upon swelling or shrinkage,may adopt a non-spherical configuration. In some cases, the particlesmay include polymeric blends. For instance, a polymer blend may beformed that includes a first polymer comprising a targeting moiety(i.e., a low-molecular weight PSMA ligand) and a biocompatible polymer,and a second polymer comprising a biocompatible polymer but notcomprising the targeting moiety. By controlling the ratio of the firstand second polymers in the final polymer, the concentration and locationof targeting moiety in the final polymer may be readily controlled toany suitable degree.

Disclosed nanoparticles may have a characteristic dimension of less thanabout 1 micrometer, where the characteristic dimension of a particle isthe diameter of a perfect sphere having the same volume as the particle.For example, the particle can have a characteristic dimension of theparticle can be less than about 300 nm, less than about 200 nm, lessthan about 150 nm, less than about 100 nm, less than about 50 nm, lessthan about 30 nm, less than about 10 nm, less than about 3 nm, or lessthan about 1 nm in some cases. In particular embodiments, thenanoparticle of the present invention has a diameter of about 80 nm-200nm, about 60 nm to about 150 nm, or about 70 nm to about 200 nm.

In one set of embodiments, the particles can have an interior and asurface, where the surface has a composition different from theinterior, i.e., there may be at least one compound present in theinterior but not present on the surface (or vice versa), and/or at leastone compound is present in the interior and on the surface at differingconcentrations. For example, in one embodiment, a compound, such as atargeting moiety (i.e., a low-molecular weight ligand) of a polymericconjugate of the present invention, may be present in both the interiorand the surface of the particle, but at a higher concentration on thesurface than in the interior of the particle, although in some cases,the concentration in the interior of the particle may be essentiallynonzero, i.e., there is a detectable amount of the compound present inthe interior of the particle.

In some cases, the interior of the particle is more hydrophobic than thesurface of the particle. For instance, the interior of the particle maybe relatively hydrophobic with respect to the surface of the particle,and a drug or other payload may be hydrophobic, and readily associateswith the relatively hydrophobic center of the particle. The drug orother payload can thus be contained within the interior of the particle,which can shelter it from the external environment surrounding theparticle (or vice versa). For instance, a drug or other payloadcontained within a particle administered to a subject will be protectedfrom a subject's body, and the body will also be isolated from the drug.Yet another aspect of the invention is directed to polymer particleshaving more than one polymer or macromolecule present, and librariesinvolving such polymers or macromolecules. For example, in one set ofembodiments, particles may contain more than one distinguishablepolymers (e.g., copolymers, e.g., block copolymers), and the ratios ofthe two (or more) polymers may be independently controlled, which allowsfor the control of properties of the particle. For instance, a firstpolymer may be a polymeric conjugate comprising a targeting moiety and abiocompatible portion, and a second polymer may comprise a biocompatibleportion but not contain the targeting moiety, or the second polymer maycontain a distinguishable biocompatible portion from the first polymer.Control of the amounts of these polymers within the polymeric particlemay thus be used to control various physical, biological, or chemicalproperties of the particle, for instance, the size of the particle(e.g., by varying the molecular weights of one or both polymers), thesurface charge (e.g., by controlling the ratios of the polymers if thepolymers have different charges or terminal groups), the surfacehydrophilicity (e.g., if the polymers have different molecular weightsand/or hydrophilicities), the surface density of the targeting moiety(e.g., by controlling the ratios of the two or more polymers), etc.

As a specific example, a particle can comprise a first diblock polymercomprising a poly(ethylene glycol) and a targeting moiety conjugated tothe poly(ethylene glycol), and a second polymer comprising thepoly(ethylene glycol) but not the targeting moiety, or comprising boththe poly(ethylene glycol) and the targeting moiety, where thepoly(ethylene glycol) of the second polymer has a different length (ornumber of repeat units) than the poly(ethylene glycol) of the firstpolymer. As another example, a particle may comprise a first polymercomprising a first biocompatible portion and a targeting moiety, and asecond polymer comprising a second biocompatible portion different fromthe first biocompatible portion (e.g., having a different composition, asubstantially different number of repeat units, etc.) and the targetingmoiety. As yet another example, a first polymer may comprise abiocompatible portion and a first targeting moiety, and a second polymermay comprise a biocompatible portion and a second targeting moietydifferent from the first targeting moiety.

For example, disclosed herein is a therapeutic polymeric nanoparticlecapable of binding to a target, comprising a first non-functionalizedpolymer; an optional second non-functionalized polymer; a functionalizedpolymer comprising a targeting moiety; and a therapeutic agent; whereinsaid nanoparticle comprises about 15 to about 300 molecules offunctionalized polymer, or about 20 to about 200 molecule, or about 3 toabout 100 molecules of functionalized polymer.

In a particular embodiment, the polymer of the first or secondmacromolecules of the nanoparticle of the invention is PLA, PLGA, orPEG, or copolymers thereof. In a specific embodiment, the polymer of thefirst macromolecule is a PLGA-PEG copolymer, and the secondmacromolecule is a PLGA-PEG copolymer, or a PLA-PEG copolymer. Forexample, exemplary nanoparticle may have a PEG corona with a density ofabout 0.065 g/cm³, or about 0.01 to about 0.10 g/cm³.

Disclosed nanoparticles may be stable (e.g. retain substantially allactive agent) for example in a solution that may contain a saccharide,for at least about 3 days, about 4 days or at least about 5 days at roomtemperature, or at 25° C.

In some embodiments, disclosed nanoparticles may also include a fattyalcohol, which may increase the rate of drug release. For example,disclosed nanoparticles may include a C₈-C₃₀ alcohol such as cetylalcohol, octanol, stearyl alcohol, arachidyl alcohol, docosonal, oroctasonal.

Nanoparticles may have controlled release properties, e.g., may becapable of delivering an amount of active agent to a patient, e.g., tospecific site in a patient, over an extended period of time, e.g. over 1day, 1 week, or more. In some embodiments, disclosed nanoparticlessubstantially immediately releases (e.g. over about 1 minute to about 30minutes) less than about 2%, less than about 5%, or less than about 10%of an active agent (e.g. a taxane) agent, for example when places in aphosphate buffer solution at room temperature and/or at 37° C.

For example, disclosed nanoparticles that include a therapeutic agent,may, in some embodiments, may release the therapeutic agent when placedin an aqueous solution at e.g., 25 C with a rate substantiallycorresponding to a) from about 0.01 to about 20% of the totaltherapeutic agent is released after about 1 hour; b) from about 10 toabout 60% of the therapeutic agent is released after about 8 hours; c)from about 30 to about 80% of the total therapeutic agent is releasedafter about 12 hours; and d) not less than about 75% of the total isreleased after about 24 hours.

In some embodiments, after administration to a subject or patient of adisclosed nanoparticle or a composition that includes a disclosednanoparticle, the peak plasma concentration (C_(max)) of the therapeuticagent in the patient s substantially higher as compared to a C_(max) ofthe therapeutic agent if administered alone (e.g., not as part of ananoparticle).

In another embodiment, a disclosed nanoparticle including a therapeuticagent, when administered to a subject, may have a t_(max) of therapeuticagent substantially longer as compared to a t_(max) of the therapeuticagent administered alone.

Libraries of such particles may also be formed. For example, by varyingthe ratios of the two (or more) polymers within the particle, theselibraries can be useful for screening tests, high-throughput assays, orthe like. Entities within the library may vary by properties such asthose described above, and in some cases, more than one property of theparticles may be varied within the library. Accordingly, one embodimentof the invention is directed to a library of nanoparticles havingdifferent ratios of polymers with differing properties. The library mayinclude any suitable ratio(s) of the polymers.

FIG. 1 illustrates that libraries can be produced using polymers such asthose described above. For example, in FIG. 1, polymeric particlescomprising a first macromolecule comprising a biocompatible hydrophobicpolymer, a biocompatible hydrophilic polymer, and a low-molecular weightPSMA ligand, and a second macromolecule that comprises a biocompatiblehydrophobic polymer and a biocompatible hydrophilic polymer may be usedto create a library of particles having different ratios of the firstand second macromolecules.

Such a library may be useful in achieving particles having any number ofdesirable properties, for instance properties such as surfacefunctionality, surface charge, size, zeta (ζ) potential, hydrophobicity,ability to control immunogenicity, or the like.

As specific examples, in some embodiments of the present invention, thelibrary includes particles comprising polymeric conjugates of abiocompatible polymer and a low-molecular weight ligand, as discussedherein. Referring now to FIG. 1, one such particle is shown as anon-limiting example. In this figure, a polymeric conjugate of thedisclosure is used to form a particle 10. The polymer forming particle10 includes a low-molecular weight ligand 15, present on the surface ofthe particle, and a biocompatible portion 17. In some cases, as shownhere, targeting moiety 15 may be conjugated to biocompatible portion 17.However, not all of biocompatible portion 17 is shown conjugated totargeting moiety 15. For instance, in some cases, particles such asparticle 10 may be formed using a first polymer comprising biocompatibleportion 17 and low-molecular weight ligand 15, and a second polymercomprising biocompatible portion 17 but not targeting moiety 15. Bycontrolling the ratio of the first and second polymers, particles havingdifferent properties may be formed, and in some cases, libraries of suchparticles may be formed. In addition, contained within the center ofparticle 10 is drug 12. In some cases, drug 12 may be contained withinthe particle due to hydrophobic effects. For instance, the interior ofthe particle may be relatively hydrophobic with respect to the surfaceof the particle, and the drug may be a hydrophobic drug that associateswith the relatively hydrophobic center of the particle. In oneembodiment, the therapeutic agent is associated with the surface of,encapsulated within, surrounded by, or dispersed throughout thenanoparticle. In another embodiment, the therapeutic agent isencapsulated within the hydrophobic core of the nanoparticle.

As a specific example, particle 10 may contain polymers including arelatively hydrophobic biocompatible polymer and a relativelyhydrophilic targeting moiety 15, such that, during particle formation, agreater concentration of the hydrophilic targeting moiety is exposed onthe surface and a greater concentration of the hydrophobic biocompatiblepolymer is present within the interior of the particle.

In some embodiments, the biocompatible polymer is a hydrophobic polymer.Non-limiting examples of biocompatible polymers include polylactide,polyglycolide, and/or poly(lactide-co-glycolide).

In a different embodiment, this disclosure provides for a nanoparticlecomprising 1) a polymeric matrix; 2) optionally, an amphiphilic compoundor layer that surrounds or is dispersed within the polymeric matrixforming a continuous or discontinuous shell for the particle; 3) anon-functionalized polymer that may form part of the polymeric matrix,and 4) a low molecular weight PSMA ligand covalently attached to apolymer, which may form part of the polymeric matrix. For example, anamphiphilic layer may reduce water penetration into the nanoparticle,thereby enhancing drug encapsulation efficiency and slowing drugrelease.

As used herein, the term “amphiphilic” refers to a property where amolecule has both a polar portion and a non-polar portion. Often, anamphiphilic compound has a polar head attached to a long hydrophobictail. In some embodiments, the polar portion is soluble in water, whilethe non-polar portion is insoluble in water. In addition, the polarportion may have either a formal positive charge, or a formal negativecharge. Alternatively, the polar portion may have both a formal positiveand a negative charge, and be a zwitterion or inner salt. For purposesof the invention, the amphiphilic compound can be, but is not limitedto, one or a plurality of the following: naturally derived lipids,surfactants, or synthesized compounds with both hydrophilic andhydrophobic moieties.

Specific examples of amphiphilic compounds include, but are not limitedto, phospholipids, such as 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), diarachidoylphosphatidylcholine (DAPC),dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine(DTPC), and dilignoceroylphatidylcholine (DLPC), incorporated at a ratioof between 0.01-60 (weight lipid/w polymer), most preferably between0.1-30 (weight lipid/w polymer). Phospholipids which may be usedinclude, but are not limited to, phosphatidic acids, phosphatidylcholines with both saturated and unsaturated lipids, phosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines,phosphatidylinositols, lysophosphatidyl derivatives, cardiolipin, andβ-acyl-y-alkyl phospholipids. Examples of phospholipids include, but arenot limited to, phosphatidylcholines such asdioleoylphosphatidylcholine, dimyristoylphosphatidylcholine,dipentadecanoylphosphatidylcholine dilauroylphosphatidylcholine,dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), diarachidoylphosphatidylcholine (DAPC),dibehenoylphosphatidylcho-line (DBPC), ditricosanoylphosphatidylcholine(DTPC), dilignoceroylphatidylcholine (DLPC); andphosphatidylethanolamines such as dioleoylphosphatidylethanolamine or1-hexadecyl-2-palmitoylglycerophos-phoethanolamine. Syntheticphospholipids with asymmetric acyl chains (e.g., with one acyl chain of6 carbons and another acyl chain of 12 carbons) may also be used.

In a particular embodiment, an amphiphilic component that can be used toform an amphiphilic layer is lecithin, and, in particular,phosphatidylcholine. Lecithin is an amphiphilic lipid and, as such,forms a phospholipid bilayer having the hydrophilic (polar) heads facingtheir surroundings, which are oftentimes aqueous, and the hydrophobictails facing each other. Lecithin has an advantage of being a naturallipid that is available from, e.g., soybean, and already has FDAapproval for use in other delivery devices. In addition, a mixture oflipids such as lethicin is more advantageous than one single pure lipid.

In certain embodiments a disclosed nanoparticle has an amphiphilicmonolayer, meaning the layer is not a phospholipid bilayer, but existsas a single continuous or discontinuous layer around, or within, thenanoparticle. The amphiphilic layer is “associated with” thenanoparticle of the invention, meaning it is positioned in someproximity to the polymeric matrix, such as surrounding the outside ofthe polymeric shell, or dispersed within the polymers that make up thenanoparticle.

Preparation of Nanoparticles

Another aspect of this disclosure is directed to systems and methods ofmaking disclosed nanoparticles. In some embodiments, using two or moredifferent polymers (e.g., copolymers, e.g., block copolymers) indifferent ratios and producing particles from the polymers (e.g.,copolymers, e.g., block copolymers), properties of the particles becontrolled. For example, one polymer (e.g., copolymer, e.g., blockcopolymer) may or may not include a low-molecular weight PSMA ligand,while another optional polymer (e.g., copolymer, e.g., block copolymer)may be chosen for its biocompatibility and/or its ability to controlimmunogenicity of the resultant particle.

In one set of embodiments, the particles are formed by providing asolution comprising one or more polymers, and contacting the solutionwith a polymer nonsolvent to produce the particle. The solution may bemiscible or immiscible with the polymer nonsolvent. For example, awater-miscible liquid such as acetonitrile may contain the polymers, andparticles are formed as the acetonitrile is contacted with water, apolymer nonsolvent, e.g., by pouring the acetonitrile into the water ata controlled rate. The polymer contained within the solution, uponcontact with the polymer nonsolvent, may then precipitate to formparticles such as nanoparticles. Two liquids are said to be “immiscible”or not miscible, with each other when one is not soluble in the other toa level of at least 10% by weight at ambient temperature and pressure.Typically, an organic solution (e.g., dichloromethane, acetonitrile,chloroform, tetrahydrofuran, acetone, formamide, dimethylformamide,pyridines, dioxane, dimethysulfoxide, etc.) and an aqueous liquid (e.g.,water, or water containing dissolved salts or other species, cell orbiological media, ethanol, etc.) are immiscible with respect to eachother. For example, the first solution may be poured into the secondsolution (at a suitable rate or speed). In some cases, particles such asnanoparticles may be formed as the first solution contacts theimmiscible second liquid, e.g., precipitation of the polymer uponcontact causes the polymer to form nanoparticles while the firstsolution poured into the second liquid, and in some cases, for example,when the rate of introduction is carefully controlled and kept at arelatively slow rate, nanoparticles may form. The control of suchparticle formation can be readily optimized by one of ordinary skill inthe art using only routine experimentation.

Properties such as surface functionality, surface charge, size, zeta (ζ)potential, hydrophobicity, ability to control immunogenicity, and thelike, may be highly controlled using a disclosed process. For instance,a library of particles may be synthesized, and screened to identify theparticles having a particular ratio of polymers that allows theparticles to have a specific density of moieties (e.g., low-molecularweight PSMA ligands) present on the surface of the particle. This allowsparticles having one or more specific properties to be prepared, forexample, a specific size and a specific surface density of moieties,without an undue degree of effort. Accordingly, certain embodiments ofthe invention are directed to screening techniques using such libraries,as well as any particles identified using such libraries. In addition,identification may occur by any suitable method. For instance, theidentification may be direct or indirect, or proceed quantitatively orqualitatively.

In some embodiments, already-formed nanoparticles are functionalizedwith a targeting moiety using procedures analogous to those describedfor producing ligand-functionalized polymeric conjugates. For example, afirst copolymer (PLGA-PEG, poly(lactide-co-glycolide) and poly(ethyleneglycol)) is mixed with a therapeutic agent to form particles. Theparticles are then associated with a low-molecular weight ligand to formnanoparticles that can be used for the treatment of cancer. Theparticles can be associated with varying amounts of low-molecular weightligands in order to control the ligand surface density of thenanoparticle, thereby altering the therapeutic characteristics of thenanoparticle. Furthermore, for example, by controlling parameters suchas molecular weight, the molecular weight of PEG, and the nanoparticlesurface charge, very precisely controlled particles may be obtained.

In another embodiment, a nanoemulsion process is provided, such as theprocess represented in FIGS. 3 and 4. For example, a therapeutic agent,a first polymer (for example, a diblock co-polymer such as PLA-PEG orPLGA-PEG, either of which may be optionally bound to a ligand, e.g.,GL2) and an optional second polymer (e.g. (PL(G)A-PEG or PLA), with anorganic solution to form a first organic phase. Such first phase mayinclude about 5 to about 50% weight solids, e.g about 5 to about 40%solids, or about 10 to about 30% solids. The first organic phase may becombined with a first aqueous solution to form a second phase. Theorganic solution can include, for example, toluene, methyl ethyl ketone,acetonitrile, tetrahydrofuran, ethyl acetate, isopropyl alcohol,isopropyl acetate, dimethylformamide, methylene chloride,dichloromethane, chloroform, acetone, benzyl alcohol, Tween 80, Span 80,or the like, and combinations thereof. In an embodiment, the organicphase may include benzyl alcohol, ethyl acetate, and combinationsthereof. The second phase can be between about 1 and 50 weight %, e.g.,about 5-40 weight %, solids. The aqueous solution can be water,optionally in combination with one or more of sodium cholate, ethylacetate, polyvinyl acetate and benzyl alcohol.

For example, the oil or organic phase may use solvent that is onlypartially miscible with the nonsolvent (water). Therefore, when mixed ata low enough ratio and/or when using water pre-saturated with theorganic solvents, the oil phase remains liquid. The oil phase may beeemulsified into an aqueous solution and, as liquid droplets, shearedinto nanoparticles using, for example, high energy dispersion systems,such as homogenizers or sonicators. The aqueous portion of the emulsion,otherwise known as the “water phase”, may be surfactant solutionconsisting of sodium cholate and pre-saturated with ethyl acetate andbenzyl alcohol.

Emulsifying the second phase to form an emulsion phase may be performedin one or two emulsification steps. For example, a primary emulsion maybe prepared, and then emulsified to form a fine emulsion. The primaryemulsion can be formed, for example, using simple mixing, a highpressure homogenizer, probe sonicator, stir bar, or a rotor statorhomogenizer. The primary emulsion may be formed into a fine emulsionthrough the use of e.g. probe sonicator or a high pressure homogenizer,e.g. by using 1, 2, 3 or more passes through a homogenizer. For example,when a high pressure homogenizer is used, the pressure used may be about1000 to about 8000 psi, about 2000 to about 4000 psi 4000 to about 8000psi, or about 4000 to about 5000 psi, e.g., about 2000, 2500, 4000 or5000 psi.

Either solvent evaporation or dilution may be needed to complete theextraction of the solvent and solidify the particles. For better controlover the kinetics of extraction and a more scalable process, a solventdilution via aqueous quench may be used. For example, the emulsion canbe diluted into cold water to a concentration sufficient to dissolve allof the organic solvent to form a quenched phase. Quenching may beperformed at least partially at a temperature of about 5° C. or less.For example, water used in the quenching may be at a temperature that isless that room temperature (e.g. about 0 to about 10° C., or about 0 toabout 5° C.).

In some embodiments, not all of the therapeutic agent (e.g. docetaxel)is encapsulated in the particles at this stage, and a drug solubilizeris added to the quenched phase to form a solubilized phase. The drugsolubilizer may be for example, Tween 80, Tween 20, polyvinylpyrrolidone, cyclodextran, sodium dodecyl sulfate, or sodium cholate.For example, Tween-80 may added to the quenched nanoparticle suspensionto solubilize the free drug and prevent the formation of drug crystals.In some embodiments, a ratio of drug solubilizer to therapeutic agent(e.g. docetaxel) is about 100:1 to about 10:1.

The solubilized phase may be filtered to recover the nanoparticles. Forexample, ultrafiltration membranes may be used to concentrate thenanoparticle suspension and substantially eliminate organic solvent,free drug, and other processing aids (surfactants). Exemplary filtrationmay be performed using a tangential flow filtration system. For example,by using a membrane with a pore size suitable to retain nanoparticleswhile allowing solutes, micelles, and organic solvent to pass,nanoparticles can be selectively separated. Exemplary membranes withmolecular weight cut-offs of about 300-500 kDa (˜5-25 nm) may be used.

Diafiltration may be performed using a constant volume approach, meaningthe diafiltrate (cold deionized water, e.g. about 0 to about 5° C., or 0to about 10° C.) may added to the feed suspension at the same rate asthe filtrate is removed from the suspension. In some embodiments,filtering may include a first filtering using a first temperature ofabout 0 to about 5° C., or 0 to about 10° C., and a second temperatureof about 20 to about 30° C., or 15 to about 35° C. For example,filtering may include processing about 1 to about 6 diavolumes at about0 to about 5° C., and processing at least one diavolume (e.g. about 1 toabout 3 or about 1-2 diavolumes) at about 20 to about 30° C.

After purifying and concentrating the nanoparticle suspension, theparticles may be passed through one, two or more sterilizing and/ordepth filters, for example, using ˜0.2 μm depth pre-filter.

In another embodiment of preparing nanoparticles, an organic phase isformed composed of a mixture of a therapeutic agent, e.g., docetaxel,and polymer (homopolymer, co-polymer, and co-polymer with ligand). Theorganic phase is mixed with an aqueous phase at approximately a 1:5ratio (oil phase:aqueous phase) where the aqueous phase is composed of asurfactant and some dissolved solvent. The primary emulsion is formed bythe combination of the two phases under simple mixing or through the useof a rotor stator homogenizer. The primary emulsion is then formed intoa fine emulsion through the use of a high pressure homogenizer. The fineemulsion is then quenched by addition to deionized water under mixing.The quench:emulsion ratio is approximately 8.5:1. Then a solution ofTween (e.g., Tween 80) is added to the quench to achieve approximately2% Tween overall. This serves to dissolve free, unencapsulated drug. Thenanoparticles are then isolated through either centrifugation orultrafiltration/diafiltration.

It will be appreciated that the amounts of polymer and therapeutic oractive agent that are used in the preparation of the formulation maydiffer from a final formulation. For example, some active agent may notbecome completely incorporated in a nanoparticle and such freetherapeutic agent may be e.g. filtered away. For example, in anembodiment, about 20 weight percent of active agent (e.g. docetaxel) andabout 80 weight percent polymer (e.g. the polymer may include about 2.5mol percent PLA-PEG-GL2 and about 97.5 mol percent PLA-PEG). may be usedin the preparation of a formulation that results in an e.g. finalnanoparticle comprising about 10 weight percent active agent (e.g.docetaxel) and about 90 weight percent polymer (where the polymer mayinclude about 1.25 mol percent PLA-PEG-GL2 and about 98.75 mol percentPLA-PEG). Such processes may provide final nanoparticles suitable foradministration to a patient that includes about 2 to about 20 percent byweight therapeutic agent, e.g. about 5, about 8, about 10, about 15percent therapeutic agent by weight.

Therapeutic Agents

According to the present invention, any agents including, for example,therapeutic agents (e.g. anti-cancer agents), diagnostic agents (e.g.contrast agents; radionuclides; and fluorescent, luminescent, andmagnetic moieties), prophylactic agents (e.g. vaccines), and/ornutraceutical agents (e.g. vitamins, minerals, etc.) may be delivered bythe disclosed nanoparticles. Exemplary agents to be delivered inaccordance with the present invention include, but are not limited to,small molecules (e.g. cytotoxic agents), nucleic acids (e.g., siRNA,RNAi, and mircoRNA agents), proteins (e.g. antibodies), peptides,lipids, carbohydrates, hormones, metals, radioactive elements andcompounds, drugs, vaccines, immunological agents, etc., and/orcombinations thereof. In some embodiments, the agent to be delivered isan agent useful in the treatment of cancer (e.g., prostate cancer).

For instance, a targeting moiety, if used, may target or cause theparticle to become localized at specific portions within a subject, andthe payload may be delivered to those portions. In a particularembodiment, the drug or other payload may is released in a controlledrelease manner from the particle and allowed to interact locally withthe particular targeting site (e.g., a tumor). The term “controlledrelease” (and variants of that term) as used herein (e.g., in thecontext of “controlled-release system”) is generally meant to encompassrelease of a substance (e.g., a drug) at a selected site or otherwisecontrollable in rate, interval, and/or amount. Controlled releaseencompasses, but is not necessarily limited to, substantially continuousdelivery, patterned delivery (e.g., intermittent delivery over a periodof time that is interrupted by regular or irregular time intervals), anddelivery of a bolus of a selected substance (e.g., as a predetermined,discrete amount if a substance over a relatively short period of time(e.g., a few seconds or minutes)).

The active agent or drug may be a therapeutic agent such as anantineoplastic such as mTor inhibitors (e.g., sirolimus, temsirolimus,or everolimus), vinca alkaloids such as vincristine, a diterpenederivative or a taxane such as paclitaxel (or its derivatives such asDHA-paclitaxel or PG-paxlitaxel) or docetaxel.

In one set of embodiments, the payload is a drug or a combination ofmore than one drug. Such particles may be useful, for example, inembodiments where a targeting moiety may be used to direct a particlecontaining a drug to a particular localized location within a subject,e.g., to allow localized delivery of the drug to occur. Exemplarytherapeutic agents include chemotherapeutic agents such as doxorubicin(adriamycin), gemcitabine (gemzar), daunorubicin, procarbazine,mitomycin, cytarabine, etoposide, methotrexate, venorelbine,5-fluorouracil (5-FU), vinca alkaloids such as vinblastine orvincristine; bleomycin, paclitaxel (taxol), docetaxel (taxotere),aldesleukin, asparaginase, busulfan, carboplatin, cladribine,camptothecin, CPT-11, 10-hydroxy-7-ethylcamptothecin (SN38),dacarbazine, S-I capecitabine, ftorafur, 5′ deoxyfluorouridine, UFT,eniluracil, deoxycytidine, 5-azacytosine, 5-azadeoxycytosine,allopurinol, 2-chloroadenosine, trimetrexate, aminopterin,methylene-10-deazaminopterin (MDAM), oxaplatin, picoplatin, tetraplatin,satraplatin, platinum-DACH, ormaplatin, CI-973, JM-216, and analogsthereof, epirubicin, etoposide phosphate, 9-aminocamptothecin,10,11-methylenedioxycamptothecin, karenitecin, 9-nitrocamptothecin, TAS103, vindesine, L-phenylalanine mustard, ifosphamidemefosphamide,perfosfamide, trophosphamide carmustine, semustine, epothilones A-E,tomudex, 6-mercaptopurine, 6-thioguanine, amsacrine, etoposidephosphate, karenitecin, acyclovir, valacyclovir, ganciclovir,amantadine, rimantadine, lamivudine, zidovudine, bevacizumab,trastuzumab, rituximab, 5-Fluorouracil, and combinations thereof.

Non-limiting examples of potentially suitable drugs include anti-canceragents, including, for example, docetaxel, mitoxantrone, andmitoxantrone hydrochloride. In another embodiment, the payload may be ananti-cancer drug such as 20-epi-1, 25 dihydroxyvitamin D3, 4-ipomeanol,5-ethynyluracil, 9-dihydrotaxol, abiraterone, acivicin, aclarubicin,acodazole hydrochloride, acronine, acylfiilvene, adecypenol, adozelesin,aldesleukin, all-tk antagonists, altretamine, ambamustine, ambomycin,ametantrone acetate, amidox, amifostine, aminoglutethimide,aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole,andrographolide, angiogenesis inhibitors, antagonist D, antagonist G,antarelix, anthramycin, anti-dorsalizing morphogenetic protein-1,antiestrogen, antineoplaston, antisense oligonucleotides, aphidicolinglycinate, apoptosis gene modulators, apoptosis regulators, apurinicacid, ARA-CDP-DL-PTBA, arginine deaminase, asparaginase, asperlin,asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2,axinastatin 3, azacitidine, azasetron, azatoxin, azatyrosine, azetepa,azotomycin, baccatin III derivatives, balanol, batimastat,benzochlorins, benzodepa, benzoylstaurosporine, beta lactam derivatives,beta-alethine, betaclamycin B, betulinic acid, BFGF inhibitor,bicalutamide, bisantrene, bisantrene hydrochloride,bisazuidinylspermine, bisnafide, bisnafide dimesylate, bistratene A,bizelesin, bleomycin, bleomycin sulfate, BRC/ABL antagonists, breflate,brequinar sodium, bropirimine, budotitane, busulfan, buthioninesulfoximine, cactinomycin, calcipotriol, calphostin C, calusterone,camptothecin derivatives, canarypox IL-2, capecitabine, caraceraide,carbetimer, carboplatin, carboxamide-amino-triazole,carboxyamidotriazole, carest M3, carmustine, earn 700, cartilage derivedinhibitor, carubicin hydrochloride, carzelesin, casein kinaseinhibitors, castanosperrnine, cecropin B, cedefingol, cetrorelix,chlorambucil, chlorins, chloroquinoxaline sulfonamide, cicaprost,cirolemycin, cisplatin, cis-porphyrin, cladribine, clomifene analogs,clotrimazole, collismycin A, collismycin B, combretastatin A4,combretastatin analog, conagenin, crambescidin 816, crisnatol, crisnatolmesylate, cryptophycin 8, cryptophycin A derivatives, curacin A,cyclopentanthraquinones, cyclophosphamide, cycloplatam, cypemycin,cytarabine, cytarabine ocfosfate, cytolytic factor, cytostatin,dacarbazine, dacliximab, dactinomycin, daunorubicin hydrochloride,decitabine, dehydrodidemnin B, deslorelin, dexifosfamide, dexormaplatin,dexrazoxane, dexverapamil, dezaguanine, dezaguanine mesylate,diaziquone, didemnin B, didox, diethyhiorspermine,dihydro-5-azacytidine, dioxamycin, diphenyl spiromustine, docetaxel,docosanol, dolasetron, doxifluridine, doxorubicin, doxorubicinhydrochloride, droloxifene, droloxifene citrate, dromostanolonepropionate, dronabinol, duazomycin, duocannycin SA, ebselen, ecomustine,edatrexate, edelfosine, edrecolomab, eflomithine, eflomithinehydrochloride, elemene, elsarnitrucin, emitefur, enloplatin, enpromate,epipropidine, epirubicin, epirubicin hydrochloride, epristeride,erbulozole, erythrocyte gene therapy vector system, esorubicinhydrochloride, estramustine, estramustine analog, estramustine phosphatesodium, estrogen agonists, estrogen antagonists, etanidazole, etoposide,etoposide phosphate, etoprine, exemestane, fadrozole, fadrozolehydrochloride, fazarabine, fenretinide, filgrastim, finasteride,flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine,fludarabine phosphate, fluorodaunorunicin hydrochloride, fluorouracil,fluorocitabine, forfenimex, formestane, fosquidone, fostriecin,fostriecin sodium, fotemustine, gadolinium texaphyrin, gallium nitrate,galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, gemcitabinehydrochloride, glutathione inhibitors, hepsulfam, heregulin,hexamethylene bisacetamide, hydroxyurea, hypericin, ibandronic acid,idarubicin, idarubicin hydrochloride, idoxifene, idramantone,ifosfamide, ihnofosine, ilomastat, imidazoacridones, imiquimod,immunostimulant peptides, insulin-like growth factor-1 receptorinhibitor, interferon agonists, interferon alpha-2A, interferonalpha-2B, interferon alpha-N1, interferon alpha-N3, interferon beta-IA,interferon gamma-IB, interferons, interleukins, iobenguane,iododoxorubicin, iproplatm, irinotecan, irinotecan hydrochloride,iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron,jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide,lanreotide acetate, leinamycin, lenograstim, lentinan sulfate,leptolstatin, letrozole, leukemia inhibiting factor, leukocyte alphainterferon, leuprolide acetate, leuprolide/estrogen/progesterone,leuprorelin, levamisole, liarozole, liarozole hydrochloride, linearpolyamine analog, lipophilic disaccharide peptide, lipophilic platinumcompounds, lissoclinamide, lobaplatin, lombricine, lometrexol,lometrexol sodium, lomustine, lonidamine, losoxantrone, losoxantronehydrochloride, lovastatin, loxoribine, lurtotecan, lutetium texaphyrinlysofylline, lytic peptides, maitansine, mannostatin A, marimastat,masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinaseinhibitors, maytansine, mechlorethamine hydrochloride, megestrolacetate, melengestrol acetate, melphalan, menogaril, merbarone,mercaptopurine, meterelin, methioninase, methotrexate, methotrexatesodium, metoclopramide, metoprine, meturedepa, microalgal protein kinaseC uihibitors, MIF inhibitor, mifepristone, miltefosine, mirimostim,mismatched double stranded RNA, mitindomide, mitocarcin, mitocromin,mitogillin, mitoguazone, mitolactol, mitomalcin, mitomycin, mitomycinanalogs, mitonafide, mitosper, mitotane, mitotoxin fibroblast growthfactor-saporin, mitoxantrone, mitoxantrone hydrochloride, mofarotene,molgramostim, monoclonal antibody, human chorionic gonadotrophin,monophosphoryl lipid a/myobacterium cell wall SK, mopidamol, multipledrug resistance gene inhibitor, multiple tumor suppressor 1-basedtherapy, mustard anticancer agent, mycaperoxide B, mycobacterial cellwall extract, mycophenolic acid, myriaporone, n-acetyldinaline,nafarelin, nagrestip, naloxone/pentazocine, napavin, naphterpin,nartograstim, nedaplatin, nemorubicin, neridronic acid, neutralendopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxideantioxidant, nitrullyn, nocodazole, nogalamycin, n-substitutedbenzamides, O6-benzylguanine, octreotide, okicenone, oligonucleotides,onapristone, ondansetron, oracin, oral cytokine inducer, ormaplatin,osaterone, oxaliplatin, oxaunomycin, oxisuran, paclitaxel, paclitaxelanalogs, paclitaxel derivatives, palauamine, palmitoylrhizoxin,pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine,pegaspargase, peldesine, peliomycin, pentamustine, pentosan polysulfatesodium, pentostatin, pentrozole, peplomycin sulfate, perflubron,perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate,phosphatase inhibitors, picibanil, pilocarpine hydrochloride,pipobroman, piposulfan, pirarubicin, piritrexim, piroxantronehydrochloride, placetin A, placetin B, plasminogen activator inhibitor,platinum complex, platinum compounds, platinum-triamine complex,plicamycin, plomestane, porfimer sodium, porfiromycin, prednimustine,procarbazine hydrochloride, propyl bis-acridone, prostaglandin J2,prostatic carcinoma antiandrogen, proteasome inhibitors, protein A-basedimmune modulator, protein kinase C inhibitor, protein tyrosinephosphatase inhibitors, purine nucleoside phosphorylase inhibitors,puromycin, puromycin hydrochloride, purpurins, pyrazorurin,pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene conjugate,RAF antagonists, raltitrexed, ramosetron, RAS farnesyl proteintransferase inhibitors, RAS inhibitors, RAS-GAP inhibitor, retelliptinedemethylated, rhenium RE 186 etidronate, rhizoxin, riboprine, ribozymes,RH retinamide, RNAi, rogletimide, rohitukine, romurtide, roquinimex,rubiginone B1, ruboxyl, safingol, safingol hydrochloride, saintopin,sarcnu, sarcophytol A, sargramostim, SDI1 mimetics, semustine,senescence derived inhibitor 1, sense oligonucleotides, signaltransduction inhibitors, signal transduction modulators, simtrazene,single chain antigen binding protein, sizofuran, sobuzoxane, sodiumborocaptate, sodium phenylacetate, solverol, somatomedin bindingprotein, sonermin, sparfosafe sodium, sparfosic acid, sparsomycin,spicamycin D, spirogermanium hydrochloride, spiromustine, spiroplatin,splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-celldivision inhibitors, stipiamide, streptonigrin, streptozocin,stromelysin inhibitors, sulfinosine, sulofenur, superactive vasoactiveintestinal peptide antagonist, suradista, suramin, swainsonine,synthetic glycosaminoglycans, talisomycin, tallimustine, tamoxifenmethiodide, tauromustine, tazarotene, tecogalan sodium, tegafur,tellurapyrylium, telomerase inhibitors, teloxantrone hydrochloride,temoporfin, temozolomide, teniposide, teroxirone, testolactone,tetrachlorodecaoxide, tetrazomine, thaliblastine, thalidomide,thiamiprine, thiocoraline, thioguanine, thiotepa, thrombopoietin,thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist,thymotrinan, thyroid stimulating hormone, tiazofurin, tin ethyletiopurpurin, tirapazamine, titanocene dichloride, topotecanhydrochloride, topsentin, toremifene, toremifene citrate, totipotentstem cell factor, translation inhibitors, trestolone acetate, tretinoin,triacetyluridine, triciribine, triciribine phosphate, trimetrexate,trimetrexate glucuronate, triptorelin, tropisetron, tubulozolehydrochloride, turosteride, tyrosine kinase inhibitors, tyrphostins, UBCinhibitors, ubenimex, uracil mustard, uredepa, urogenital sinus-derivedgrowth inhibitory factor, urokinase receptor antagonists, vapreotide,variolin B, velaresol, veramine, verdins, verteporfin, vinblastinesulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidinesulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine orvinorelbine tartrate, vinrosidine sulfate, vinxaltine, vinzolidinesulfate, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb,zinostatin, zinostatin stimalamer, or zorubicin hydrochloride.

Pharmaceutical Formulations

Nanoparticles disclosed herein may be combined with pharmaceuticalacceptable carriers to form a pharmaceutical composition, according toanother aspect of the invention. As would be appreciated by one of skillin this art, the carriers may be chosen based on the route ofadministration as described below, the location of the target issue, thedrug being delivered, the time course of delivery of the drug, etc.

The pharmaceutical compositions of this invention can be administered toa patient by any means known in the art including oral and parenteralroutes. The term “patient,” as used herein, refers to humans as well asnon-humans, including, for example, mammals, birds, reptiles,amphibians, and fish. For instance, the non-humans may be mammals (e.g.,a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate,or a pig). In certain embodiments parenteral routes are desirable sincethey avoid contact with the digestive enzymes that are found in thealimentary canal. According to such embodiments, inventive compositionsmay be administered by injection (e.g., intravenous, subcutaneous orintramuscular, intraperitoneal injection), rectally, vaginally,topically (as by powders, creams, ointments, or drops), or by inhalation(as by sprays).

In a particular embodiment, the nanoparticles of the present inventionare administered to a subject in need thereof systemically, e.g., by IVinfusion or injection.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P., and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Inone embodiment, the inventive conjugate is suspended in a carrier fluidcomprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) TWEEN™80. The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, theencapsulated or unencapsulated conjugate is mixed with at least oneinert, pharmaceutically acceptable excipient or carrier such as sodiumcitrate or dicalcium phosphate and/or (a) fillers or extenders such asstarches, lactose, sucrose, glucose, mannitol, and silicic acid, (b)binders such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectantssuch as glycerol, (d) disintegrating agents such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate, (e) solution retarding agents such as paraffin,(f) absorption accelerators such as quaternary ammonium compounds, (g)wetting agents such as, for example, cetyl alcohol and glycerolmonostearate, (h) absorbents such as kaolin and bentonite clay, and (i)lubricants such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof. Inthe case of capsules, tablets, and pills, the dosage form may alsocomprise buffering agents.

It will be appreciated that the exact dosage of the PSMA-targetedparticle is chosen by the individual physician in view of the patient tobe treated, in general, dosage and administration are adjusted toprovide an effective amount of the PSMA-targeted particle to the patientbeing treated. As used herein, the “effective amount” of anPSMA-targeted particle refers to the amount necessary to elicit thedesired biological response. As will be appreciated by those of ordinaryskill in this art, the effective amount of PSMA-targeted particle mayvary depending on such factors as the desired biological endpoint, thedrug to be delivered, the target tissue, the route of administration,etc. For example, the effective amount of PSMA-targeted particlecontaining an anti-cancer drug might be the amount that results in areduction in tumor size by a desired amount over a desired period oftime. Additional factors which may be taken into account include theseverity of the disease state; age, weight and gender of the patientbeing treated; diet, time and frequency of administration; drugcombinations; reaction sensitivities; and tolerance/response to therapy

The nanoparticles of the invention may be formulated in dosage unit formfor ease of administration and uniformity of dosage. The expression“dosage unit form” as used herein refers to a physically discrete unitof nanoparticle appropriate for the patient to be treated. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention will be decided by the attending physician withinthe scope of sound medical judgment. For any nanoparticle, thetherapeutically effective dose can be estimated initially either in cellculture assays or in animal models, usually mice, rabbits, dogs, orpigs. The animal model is also used to achieve a desirable concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic efficacy and toxicity of nanoparticles can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose is therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose is lethal to 50% of the population). Thedose ratio of toxic to therapeutic effects is the therapeutic index, andit can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceutical compositionswhich exhibit large therapeutic indices may be useful in someembodiments. The data obtained from cell culture assays and animalstudies can be used in formulating a range of dosage for human use.

In an embodiment, compositions disclosed herein may include less thanabout 10 ppm of palladium, or less than about 8 ppm, or less than about6 ppm of palladium. For example, provided here is a composition thatincludes nanoparticles having a polymeric conjugate PLA-PEG-GL2 whereinthe composition has less than about 10 ppm of palladium.

In an exemplary embodiment, a pharmaceutical composition is disclosedthat includes a plurality of nanoparticles each comprising a therapeuticagent; about 0.1 to about 30 mole percent of the total polymer content,or about 0.1 to about 20 mole percent, or about 0.1 to about 10 molepercent, or about 1 to about 5 mole percent of the total polymer contentof a nanoparticle, of a first macromolecule comprising a PLGA-PEGcopolymer or PLA-PEG copolymer that is conjugated to ligand having amolecular weight between about 100 g/mol and 500 g/mol; and a secondmacromolecule comprising a PLGA-PEG copolymer or PLA-PEG copolymer,wherein the copolymer is not bound to a targeting moiety; and apharmaceutically acceptable excipient. For example, the first copolymermay have about 0.001 and 5 weight percent of the ligand with respect tototal polymer content.

In some embodiments, a composition suitable for freezing iscomtemplated, including nanoparticles disclosed herein and a solutionsuitable for freezing, e.g. a sucrose solution is added to thenanoparticle suspension. The sucrose may e.g., as a cryoprotectant toprevent the particles from aggregating upon freezing. For example,provided herein is a nanoparticle formulation comprising a plurality ofdisclosed nanoparticles, sucrose and water; wherein thenanoparticles/sucrose/water is about 3-30%/10-30%/50-90% (w/w/w) orabout 5-10%/10-15%/80-90% (w/w/w).

Methods of Treatment

In some embodiments, targeted particles in accordance with the presentinvention may be used to treat, alleviate, ameliorate, relieve, delayonset of, inhibit progression of, reduce severity of, and/or reduceincidence of one or more symptoms or features of a disease, disorder,and/or condition. In some embodiments, inventive targeted particles maybe used to treat solid tumors, e.g., cancer and/or cancer cells. Incertain embodiments, inventive targeted particles may be used to treatany cancer wherein PSMA is expressed on the surface of cancer cells orin the tumor neovasculature in a subject in need thereof, including theneovasculature of prostate or non-prostate solid tumors. Examples of thePSMA-related indication include, but are not limited to, prostatecancer, breast cancer, non-small cell lung cancer, colorectal carcinoma,and glioblastoma.

The term “cancer” includes pre-malignant as well as malignant cancers.Cancers include, but are not limited to, prostate, gastric cancer,colorectal cancer, skin cancer, e.g., melanomas or basal cellcarcinomas, lung cancer, breast cancer, cancers of the head and neck,bronchus cancer, pancreatic cancer, urinary bladder cancer, brain orcentral nervous system cancer, peripheral nervous system cancer,esophageal cancer, cancer of the oral cavity or pharynx, liver cancer,kidney cancer, testicular cancer, biliary tract cancer, small bowel orappendix cancer, salivary gland cancer, thyroid gland cancer, adrenalgland cancer, osteosarcoma, chondrosarcoma, cancer of hematologicaltissues, and the like. “Cancer cells” can be in the form of a tumor,exist alone within a subject (e.g., leukemia cells), or be cell linesderived from a cancer.

Cancer can be associated with a variety of physical symptoms. Symptomsof cancer generally depend on the type and location of the tumor. Forexample, lung cancer can cause coughing, shortness of breath, and chestpain, while colon cancer often causes diarrhea, constipation, and bloodin the stool. However, to give but a few examples, the followingsymptoms are often generally associated with many cancers: fever,chills, night sweats, cough, dyspnea, weight loss, loss of appetite,anorexia, nausea, vomiting, diarrhea, anemia, jaundice, hepatomegaly,hemoptysis, fatigue, malaise, cognitive dysfunction, depression,hormonal disturbances, neutropenia, pain, non-healing sores, enlargedlymph nodes, peripheral neuropathy, and sexual dysfunction.

In one aspect of the invention, a method for the treatment of cancer(e.g. prostate or breast cancer) is provided. In some embodiments, thetreatment of cancer comprises administering a therapeutically effectiveamount of inventive targeted particles to a subject in need thereof, insuch amounts and for such time as is necessary to achieve the desiredresult. In certain embodiments of the present invention a“therapeutically effective amount” of an inventive targeted particle isthat amount effective for treating, alleviating, ameliorating,relieving, delaying onset of, inhibiting progression of, reducingseverity of, and/or reducing incidence of one or more symptoms orfeatures of cancer.

In one aspect of the invention, a method for administering inventivecompositions to a subject suffering from cancer (e.g. prostate cancer)is provided. In some embodiments, particles to a subject in such amountsand for such time as is necessary to achieve the desired result (i.e.treatment of cancer). In certain embodiments of the present invention a“therapeutically effective amount” of an inventive targeted particle isthat amount effective for treating, alleviating, ameliorating,relieving, delaying onset of, inhibiting progression of, reducingseverity of, and/or reducing incidence of one or more symptoms orfeatures of cancer.

Inventive therapeutic protocols involve administering a therapeuticallyeffective amount of an inventive targeted particle to a healthyindividual (i.e., a subject who does not display any symptoms of cancerand/or who has not been diagnosed with cancer). For example, healthyindividuals may be “immunized” with an inventive targeted particle priorto development of cancer and/or onset of symptoms of cancer; at riskindividuals (e.g., patients who have a family history of cancer;patients carrying one or more genetic mutations associated withdevelopment of cancer; patients having a genetic polymorphism associatedwith development of cancer; patients infected by a virus associated withdevelopment of cancer; patients with habits and/or lifestyles associatedwith development of cancer; etc.) can be treated substantiallycontemporaneously with (e.g., within 48 hours, within 24 hours, orwithin 12 hours of) the onset of symptoms of cancer. Of courseindividuals known to have cancer may receive inventive treatment at anytime.

In other embodiments, the nanoparticles of the present invention can beused to inhibit the growth of cancer cells, e.g., prostate cancer cells.As used herein, the term “inhibits growth of cancer cells” or“inhibiting growth of cancer cells” refers to any slowing of the rate ofcancer cell proliferation and/or migration, arrest of cancer cellproliferation and/or migration, or killing of cancer cells, such thatthe rate of cancer cell growth is reduced in comparison with theobserved or predicted rate of growth of an untreated control cancercell. The term “inhibits growth” can also refer to a reduction in sizeor disappearance of a cancer cell or tumor, as well as to a reduction inits metastatic potential. Preferably, such an inhibition at the cellularlevel may reduce the size, deter the growth, reduce the aggressiveness,or prevent or inhibit metastasis of a cancer in a patient. Those skilledin the art can readily determine, by any of a variety of suitableindicia, whether cancer cell growth is inhibited.

Inhibition of cancer cell growth may be evidenced, for example, byarrest of cancer cells in a particular phase of the cell cycle, e.g.,arrest at the G2/M phase of the cell cycle. Inhibition of cancer cellgrowth can also be evidenced by direct or indirect measurement of cancercell or tumor size. In human cancer patients, such measurementsgenerally are made using well known imaging methods such as magneticresonance imaging, computerized axial tomography and X-rays. Cancer cellgrowth can also be determined indirectly, such as by determining thelevels of circulating carcinoembryonic antigen, prostate specificantigen or other cancer-specific antigens that are correlated withcancer cell growth. Inhibition of cancer growth is also generallycorrelated with prolonged survival and/or increased health andwell-being of the subject.

Also provided herein are methods of administering to a patient ananoparticle disclosed herein including an active agent, wherein, uponadministration to a patient, such nanoparticles substantially reducesthe volume of distribution and/or substantially reduces free Cmax, ascompared to administration of the agent alone (i.e. not as a disclosednanoparticle).

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

Example 1 Synthesis of a Low-Molecular Weight PSMA Ligand (GL2)

5 g (10.67 mmol) of the starting compound was dissolved in 150 mL ofanhydrous DMF. To this solution was added allyl bromide (6.3 mL, 72mmol) and K₂CO₃ (1.47 g, 10.67 mmol). The reaction was stirred for 2 h,the solvent was removed, the crude material was dissolved in AcOEt andwashed with H₂O until pH neutral. The organic phase was dried with MgSO₄(anhydrous) and evaporated to give 5.15 g (95%) of material. (TLC inCH₂Cl₂:MeOH 20:1 Rf=0.9, started compound Rf=0.1, revealed withninhydrin and uv light).

To a solution of the compound (5.15 g, 10.13 mmol) in CH₃CN (50 mL) wasadded Et₂NH (20 mL, 0.19 mol). The reaction was stirred at roomtemperature for 40 min. The solvent was removed and the compound waspurified by column chromatography (Hexane:AcOEt 3:2) to give 2.6 g(90%). (TLC in CH₂Cl₂:MeOH 10:1 Rf=0.4, revealed with ninhydrin (thecompound has a violet color). ¹H-NMR (CDCl₃, 300 MHz) δ 5.95-5.85 (m,1H, —CH₂CHCH₂), 5.36-5.24 (m, 2H, —CH₂CHCH₂), 4.62-4.60 (m, 3H,—CH₂CHCH₂, NHBoc), 3.46 (t, 1H, CH(Lys)), 3.11-3.07 (m, 2H, CH₂NHBoc),1.79 (bs, 2H, NH₂), 1.79-1.43 (m, 6H, 3CH₂(Lys)), 1.43 (s, 9H, Boc).

To a stirred solution of diallyl glutamate (3.96 g, 15 mmol) andtriphosgene (1.47 g, 4.95 mmol) in CH₂Cl₂ (143 mL) at −78° C. was addedEt₃N (6.4 mL, 46 mmol) in CH₂Cl₂ (28 mL). The reaction mixture wasallowed to warm to room temperature and stirred for 1.5 h. The Lysinederivative (2.6 g, 9.09 mmol) in a solution of CH₂Cl₂ (36 mL) was thenadded at −78° C. and the reaction was stirred at room temperature for 12h. The solution was diluted with CH₂Cl₂, washed twice with H₂O, driedover MgSO₄ (anh.) and purified by column chromatography (Hexane:AcOEt3:1→2:1→AcOEt) to give 4 g (82%) (TLC in CH₂Cl₂:MeOH 20:1 Rf=0.3,revealed with ninhydrin). ¹H-NMR (CDCl₃, 300 MHz) δ 5.97-5.84 (m, 3H,3-CH₂CHCH₂), 5.50 (bt, 2H, 2NHurea), 5.36-5.20 (m, 6H, 3-CH₂CHCH₂), 4.81(bs, 1H, NHBoc), 4.68-4.40 (m, 8H, 3-CH₂CHCH₂, CH(Lys), CH(glu)),3.09-3.05 (m, 2H, CH₂NHBoc), 2.52-2.39 (m, 2H, CH₂(glu.)), 2.25-2.14 and2.02-1.92 (2m, 2H, CH₂(glu.)), 1.87-1.64 (m, 4H, 2CH₂(Lys)), 1.51-1.35(m, 2H, CH₂(Lys)), 1.44 (s, 9H, Boc).

To a solution of the compound (4 g, 7.42 mmol) in dry CH₂Cl₂ (40 mL) wasadded at 0° C. TFA (9 mL). The reaction was stirred at room temperaturefor 1 h. The solvent was removed under vacuum until complete dryness, togive 4.1 g (quantitative). (TLC in CH₂Cl₂:MeOH 20:1 Rf=0.1, revealedwith ninhydrin). ¹H-NMR (CDCl₃, 300 MHz) δ 6.27-6.16 (2d, 2H, 2NHurea),5.96-5.82 (m, 3H, 3-CH₂CHCH₂), 5.35-5.20 (m, 6H, 3-CH₂CHCH₂), 4.61-4.55(m, 6H, 3-CH₂CHCH₂), 4.46-4.41 (m, 2H, CH(Lys), CH(glu)), 2.99 (m, 2H,CH₂NHBoc), 2.46 (m, 2H, CH₂(glu.)), 2.23-2.11 and 2.01-1.88 (2m, 2H,CH₂(glu.)), 1.88-1.67 (m, 4H, 2CH₂(Lys)), 1.45 (m, 2H, CH₂(Lys)).

To a solution of the compound (2 g, 3.6 mmol) in DMF (anh.) (62 mL)under argon was added Pd(PPh₃)₄ (0.7 g, 0.6 mmol) and morpholine (5.4mL, 60.7 mmol) at 0° C. The reaction was stirred at room temperature for1 h. The solvent was removed. The crude product was washed twice withCH₂Cl₂, and then solved in H₂O. To this solution was added a dilutedsolution of NaOH (0.01 N) until the pH was very basic. The solvent wasremoved under reduced pressure. The solid was washed again with CH₂Cl₂,AcOEt, and a mixture of MeOH—CH₂Cl₂ (1:1), solved in H₂O and neutralizedwith Amberlite IR-120H⁺ resin. The solvent was evaporated, and thecompound was precipitated with MeOH, to give 1 g (87%) of GL2. ¹H-NMR(D₂O, 300 MHz) δ 4.07 (m, 2H, CH(Lys), CH(glu)), 2.98 (m, 2H, CH₂NH₂),2.36 (m, 2H, CH₂(glu.)), 2.08-2.00 (m, 1H, CH₂(glu)), 1.93-1.60 (m, 5H,CH₂(glu.), 2CH₂(Lys)), 1.41 (m, 2H, CH₂(Lys)). Mass ESI: 320.47 [M+H⁺],342.42 [M+Na⁺].

Example 2 Synthesis of a Low-Molecular Weight PSMA Ligand (GL1)

130 mg (0.258 mmol) of the starting compound was dissolved in 3 mL ofDMF (anh.) To this solution was added allyl bromide (150 μL, 1.72 mmol)and K₂CO₃ (41 mg, 0.3 mmol). The reaction was stirred for 1 h, thesolvent was removed, the crude product was dissolved in AcOEt and washedwith H₂O until pH neutral. The organic phase was dried with MgSO₄ (anh.)and evaporated to give 130 mg (93%). (TLC in CH₂Cl₂:MeOH 20:1 Rf=0.9,started compound Rf=0.1, revealed with ninhydrin and uv light). ¹H-NMR(CDCl₃, 300 MHz) δ 7.81-7.05 (12H, aromatics), 6.81 (bs, 1H, NHFmoc),5.93-5.81 (m, 1H, —CH₂CHCH₂), 5.35-5.24 (m, 2H, —CH₂CHCH₂), 5.00 (bd,1H, NHboc), 4.61-4.53 (m, 5H, —CH₂CHCH₂, CH₂(Fmoc), CH(pheala.)), 4.28(t, 1H, CH(Fmoc)), 3.12-2.98 (m, 2H, CH₂(pheala.), 1.44 (s, 9H, Boc).

To a solution of the compound (120 mg, 0.221 mmol) in dry CH₂Cl₂ (2 mL)was added at 0° C. TFA (1 mL). The reaction was stirred at roomtemperature for 1 h. The solvent was removed under vacuum, water wasadded and removed again, CH₂Cl₂ was added and removed again untilcomplete dryness to give 120 mg (quantitative). (TLC in CH₂Cl₂:MeOH 20:1Rf=0.1, revealed with ninhydrin and uv light). ¹H-NMR (CDCl₃, 300 MHz) δ7.80-7.00 (13H, aromatics, NHFmoc), 5.90-5.75 (m, 1H, —CH₂CHCH₂),5.35-5.19 (m, 3H, —CH₂CHCH₂, NHboc), 4.70-4.40 (2m, 5H, —CH₂CHCH₂,CH₂(Fmoc), CH(pheala.)), 4.20 (t, 1H, CH(Fmoc)), 3.40-3.05 (m, 2H,CH((pheala.)).

To a stirred solution of diallyl glutamate (110 mg, 0.42 mmol) andtriphosgene (43 mg, 0.14 mmol) in CH₂Cl₂ (4 mL) at −78° C. was addedEt₃N (180 μL, 1.3 mmol) in CH₂Cl₂ (0.8 mL). The reaction mixture wasallowed to warm to room temperature and stirred for 1.5 h. Thephenylalanine derivative (140 mg, 0.251 mmol) in a solution of CH₂Cl₂ (1mL) and Et₃N (70 μL, 0.5 mmol) was then added at −78° C. and thereaction was stirred at room temperature for 12 h. The solution wasdiluted with CH₂Cl₂, washed twice with H₂O, dried over MgSO₄ (anh.) andpurified by column chromatography (Hexane:AcOEt 3:1) to give 100 mg(57%) (TLC in CH₂Cl₂:MeOH 20:1 Rf=0.3, revealed with ninhydrin and uvlight). ¹H-NMR (CDCl₃, 300 MHz) δ 7.80-6.95 (13H, aromatics, NHFmoc),5.98-5.82 (m, 3H, 3-CH₂CHCH₂), 5.54 (bd, 1H, NHurea), 5.43-5.19 (m, 7H,3-CH₂CHCH₂, NHurea), 4.85-4.78 (m, 1H, CH(pheala.)), 4.67-4.50 (m, 9H,3-CH₂CHCH₂, CH₂(Fmoc), CH(glu.)), 4.28 (t, 1H, CH(Fmoc)), 3.05 (d, 2H,CH₂(pheala.)), 2.53-2.33 (m, 2H, CH₂(glu.)), 2.25-2.11 and 1.98-1.80(2m, 2H, CH₂(glu.)).

To a solution of the starting material (60 mg, 0.086 mmol) in CH₃CN (1mL) was added Et₂NH (1 mL, 10 mmol). The reaction was stirred at roomtemperature for 40 min. The solvent was removed and the compound waspurified by column chromatography (Hexane:AcOEt 2:1) to give 35 mg(85%). (TLC in CH₂Cl₂:MeOH 10:1 Rf=0.5, started compound Rf=0.75,revealed with ninhydrin (the compound has a violet color) and uv light).¹H-NMR (CDCl₃, 300 MHz) δ 6.85 and 6.55 (2d, 4H, aromatics), 5.98-5.82(m, 3H, 3-CH₂CHCH₂), 5.56 (bd, 1H, NHurea), 5.44-5.18 (m, 7H,3-CH₂CHCH₂, NHurea), 4.79-4.72 (m, 1H, CH(pheala.)), 4.65-4.49 (m, 7H,3-CH₂CHCH₂, CH(glu.)), 3.64 (bs, 2H, NH₂), 3.02-2.89 (m, 2H,CH((pheala.)), 2.49-2.31 (m, 2H, CH₂(glu.)), 2.20-2.09 and 1.91-1.78(2m, 2H, CH₂(glu.)).

To a solution of the compound (50 mg, 0.105 mmol) in DMF (anh.; 1.5 mL)under argon was added Pd(PPh₃)₄ (21 mg, 0.018 mmol) and morpholine (154μL, 1.77 mmol) at 0° C. The reaction was stirred at room temperature for1 h. The solvent was removed. The crude material was washed with CH₂Cl₂twice, and dissolved in H₂O. To this solution was added a dilutedsolution of NaOH (0.01 N) until the pH was very basic. The solvent wasremoved under reduced pressure. The solid was washed again with CH₂Cl₂,AcOEt, and mixture of MeOH—CH₂Cl₂ (1:1), solved in H₂O and neutralizedwith Amberlite IR-120 H⁺ resin. The solvent was evaporated and thecompound was precipitated with MeOH, to give 25 mg (67%) of GL1. ¹H-NMR(D₂O, 300 MHz) δ 7.08 and 6.79 (2d, 4H, aromatics), 4.21 (m, 1H,CH(pheala.)), 3.90 (m, 1H, CH(glu.)), 2.99 and 2.82 (2dd, 2H,CH₂(pheala.)), 2.22-2.11 (m, 2H, CH₂(glu.)), 2.05-1.70 (2m, 2H,CH₂(glu.)). ¹³C-NMR (D₂O, 75 MHz)

176.8, 174.5, 173.9 (3 COO), 153.3 (NHCONH), 138.8 (H₂N—C(Ph)), 124.5,122.9, 110.9 (aromatics), 51.3 (CH(pheala.)), 49.8 (CH(glu.)), 31.8(CH₂(pheala.)), 28.4 and 23.6 (2CH₂-glu.)). Mass ESI: 354.19 [M+H⁺],376.23 [M+Na⁺].

Example 3 Preparation of PLA-PEG

The synthesis is accomplished by ring opening polymerization ofd,l-lactide with α-hydroxy-ω-methoxypoly(ethylene glycol) as themacro-initiator, and performed at an elevated temperature using Tin (II)2-Ethyl hexanoate as a catalyst, as shown below (PEG Mn≈5,000 Da; PLAMn≈16,000 Da; PEG-PLA M_(n)≈21,000 Da)

The polymer is purified by dissolving the polymer in dichloromethane,and precipitating it in a mixture of hexane and diethyl ether. Thepolymer recovered from this step shall be dried in an oven.

Example 4 PLA-PEG-Ligand Preparation

The synthesis, shown in FIG. 2, starts with the conversion of FMOC, BOClysine to FMOC, BOC, Allyl lysine by reacting the FMOC, BOC lysine withallyl bromide and potassium carbonate in dimethyl formamide, followed bytreatment with diethyl amine in acetonitrile. The BOC, Allyl lysine isthen reacted with triphosgene and diallyl glutamate, followed bytreatment with trifluoracetic acid in methylene chloride to form thecompound “GL2P”.

The side chain amine of lysine in the GL2P is then pegylated by theaddition of Hydroxyl-PEG-Carboxylic acid with EDC and NHS. Theconjugation of GL2P to PEG is via an amide linkage. The structure ofthis resulting compound is labeled “HO-PEG-GL2P”. Following thepegylation, ring opening polymerization (ROP) of d,l-lactide with thehydroxyl group in the HO-PEG-GL2P as initiator is used to attach apolylactide block polymer to HO-PEG-GL2P via an ester bond yielding“PLA-PEG-GL2P”. Tin (II) 2-Ethyl hexanoate is used as a catalyst for thering opening polymerization.

Lastly, the allyl groups on the PLA-PEG-GL2P are removed usingmorpholine and tetrakis(triphenylphosphine) palladium (as catalyst) indichloromethane, to yield the final product PLA-PEG-Ligand. The finalcompound is purified by precipitation in 30/70% (v/v) diethylether/hexane.

Example 5 Nanoparticle Preparation—Nanoprecipitation

Nanoparticles can be prepared using GL1 or GL2 ligand. The urea-basedPSMA inhibitor GL2, which has a free amino group located in a region notcritical for PSMA binding, is synthesized from commercially availablestarting materials Boc-Phe(4NHFmoc)-OH and diallyl glutamic acid inaccordance with the procedure shown in Scheme 1. Nanoparticles areformed using nanoprecipitation: The polymer ligand conjugate isdissolved in a water miscible organic solvent together with a drug otheragent for tracking particle uptake. Additional non-functionalizedpolymer can be included to modulate the ligand surface density. Thepolymer solution is dispersed in an aqueous phase and the resultingparticles are collected by filtration. The particles can be dried orimmediately tested for cell uptake in vitro or anti-prostate tumoractivity in vivo.

Example 6 Nanoparticle Preparation—Emulsion Process

An organic phase is formed composed of 5% solids (wt %) including 2%poly(lactide-co-glycolide)-poly(ethylene glycol) diblock copolymer(PLGA-PEG; 45 kDa-5 kDa), 2% poly(D,L-lactide) (PLA; 8.5 kDa), and 1%docetaxel (DTXL) wherein docetaxel has the structure

The organic solvents are ethyl acetate (EA) and benzyl alcohol (BA)where BA comprises 20% (wt %) of the organic phase. BA is used in partto solubilize the docetaxel. The organic phase is mixed with an aqueousphase at approximately a 1:5 ratio (oil phase:aqueous phase) where theaqueous phase is composed of 0.5% sodium cholate, 2% BA, and 4% EA (wt%) in water. The primary emulsion is formed by the combination of thetwo phases under simple mixing or through the use of a rotor statorhomogenizer. The primary emulsion is then formed into a fine emulsionthrough the use of a probe sonicator or a high pressure homogenizer.

The fine emulsion is then quenched by addition to a chilled quench (0-5°C.) of deionized water under mixing. The quench:emulsion ratio isapproximately 8.5:1. Then a solution of 25% (wt %) of Tween 80 is addedto the quench to achieve approximately 2% Tween 80 overall. Thenanoparticles are then isolated through either centrifugation orultrafiltration/diafiltration. The nanoparticle suspension may then befrozen with a cyroprotectant, such as 10 wt % sucrose.

The addition of PLA in addition to the PLGA-PEG copolymer was found tosignificantly increase the drug load. It is possible the use of BAitself also serves to increase the encapsulation efficiency as well,increasing the encapsulation efficiency even if the BA was not requiredto solubilize the DTXL. The temperature of the quench was found to playa critical role in drug loading. The use of a cold quench (generallymaintained at 0-5° C.) significantly increased the drug loading comparedto the drug loading when a room temperature quench was used.

DTXL has very low water solubility, and it was found that unencapsulatedDTXL often formed crystals which were difficult to isolate from theformed nanoparticles. Drug solubilizer (Tween 80) was added after thefine emulsion has been quenched. Tween 80 is able to effectivelysolubilize DTXL crystals and allow for the isolation of nanoparticlesfrom unencapsulated DTXL by preventing the formation of DTXL crystals,and/or by effectively solubilizing any DTXL crystals that formed whenthe fine emulsion is quenched A standard set of nanoemulsion conditionswere as follows:

Control:

Attribute Value Block co-polymer (type/amount) 45/5 PLGA (50/50 L:G)-PEG(5 Kda), 80% Homopolymer (type/amount) None Drug (amount DTXL) 10%Organic solvent (type/amount) Ethyl acetate (EA) Organic cosolvent(type/amount) None Water phase 1% PVA with 6.5% EA Quench temperature~5° C. RESULTS Particle size 191 nm Drug load 0.8% In vitro release (24hour Not determined (ND) at 37 C.) Other NA

The addition of homopolymer as an additive yielded increased drug loadwhile particle size is decreased, as shown below:

Attribute Control value Example value Block copolymer 45/5 PLGA (50/50L:G)- 45/5 PLGA (50/50 L:G)- (type/amount) PEG (5 kDa, 90% PEG (5 kDa),45% Homopolymer None 8.5 kDa PLA, 45% (type/amount) Drug (amount 10% 10%DTXL) Organic solvent EA EA, 80% (type/amount) Organic cosolvent NoneBenzyl alcohol (BA), 20% (type/amount) Water phase 1% PVA with 6.5% EA1% PVA with 6.5% EA Quench ~5° C. ~5° C. temperature RESULTS Particlesize 191 nm 134 nm Drug load 0.8% 2.4% In vitro release Not determined(ND) Not determined (ND) (24 hour at 37 C.) Other NA NAQuench Temperature

Here the control used for comparison is different from the controlabove, as those were already performed at cold quench temperature.

Attribute Control value Example value Block copolymer 45/5 PLGA (50/50L:G)- 45/5 PLGA (50/50 L:G), (type/amount) PEG (47.5% 47.5% Homopolymer~30 kDa PLGA (50/50 ~30 kDa PLGA (type/amount) L:G), 47.5% (50/50 L:G),47.5% Drug (amount 5% 5% DTXL) Organic solvent Dichloromethane (DCM)Dichloromethane (DCM) (type/amount) Organic cosolvent None None(type/amount) Water phase 0.5% sodium cholate 0.5% sodium cholate Quenchtemperature ~25° C. ~5° C. RESULTS Particle size 210 nm 214 nm Drug load1.2% 3.6% In vitro release (24 Not determined (ND) Not determined (ND)hour at 37 C.) Other NA NAExemplary Parameters

Attribute Value Block copolymer (type/amount) 45/5 PLA-PEG, 40% (5 mol %containing PLA-PEG-GL2) Homopolymer (type/amount) 8.5 kDa PLA, 40% Drug(amount DTXL) 20% Organic solvent (type/amount) EA, 80% Organiccosolvent (type/amount) BA, 20% Water phase 0.5% sodium cholate, 4% EA,2% BA Quench temperature ~5° C. RESULTS Particle size 98.5 nm Drug load3.0% In vitro release (24 hour at 37 C.) ~60%

Example 7 Emulsion Process

The process described below uses an increase in the solids content ofthe oil phase. A general flow chart of the process is depicted in FIG.3, and a process flow diagram is depicted in FIG. 4. By reducing thesolvent content of the emulsified oil phase, less drug is lost to thequench fluid when the nanoparticles are hardened. A solids and solventsystem are chosen to avoid being overly viscous, which may limit theability to emulsify into ˜100 nm droplets. The use of a relatively lowmolecular weight copolymer (PLA-PEG of ˜16 kDa-5 kDa) and low molecularweight homopolymer (PLA of ˜7 kDa) allows the formulation to remain oflow enough viscosity at high solids content. A solvent system is chosenhaving a suitable solvating power to keep the drug in solution at highconcentrations. Use of a co-solvent system (typically 79:21 ethylacetate:benzyl alcohol) allows for a continuous solution up to 50%solids with an 80:20 polymer:docetaxel blend.

An organic phase is formed composed of a mixture of docetaxel (DTXL) andpolymer (homopolymer, co-polymer, and co-polymer with ligand). Theorganic phase is mixed with an aqueous phase at approximately a 1:5ratio (oil phase:aqueous phase) where the aqueous phase is composed of asurfactant and some dissolved solvent. In order to achieve high drugloading, about 30% solids in the organic phase is used.

An organic phase is formed composed of a mixture of docetaxel (DTXL) andpolymer (homopolymer, co-polymer, and co-polymer with ligand).Compositions and organic solvents are listed on the table. The organicphase is mixed with an aqueous phase at approximately a 1:5 ratio (oilphase:aqueous phase) where the aqueous phase is composed of a surfactantand some dissolved solvent. The primary emulsion is formed by thecombination of the two phases under simple mixing or through the use ofa rotor stator homogenizer. The primary emulsion is then formed into afine emulsion through the use of a high pressure homogenizer. The fineemulsion is then quenched by addition to deionized water at a giventemperature (listed on table) under mixing. The quench:emulsion ratio isapproximately 8.5:1. Then a solution of 25% (wt %) of Tween 80 is addedto the quench to achieve approximately 2% Tween 80 overall. This servesto dissolve free, unencapsulated drug, and makes the nanoparticleisolation process feasible. The nanoparticles are then isolated througheither centrifugation or ultrafiltration/diafiltration.

Control

A standard set of nanoemulsion conditions are provided as follows.Non-ligand containing particles (non-targeted nanoparticles) are formed.

Attribute Value Lot # 15-157D Homopolymer (type/amount) 6.5 kDa PLACopolymer (type/amount) 16/5 PLA-PEG, 40% Drug (amount DTXL) 20% Organicsolvent (type/amount) Ethyl acetate (EA), 79% Organic cosolvent(type/amount) Benzyl alcohol (BA), 21% Water phase 0.5% sodium cholate,2% BA, 4% EA in water [solids] in oil phase 5 wt % RESULTS Particle size114.7 nm Drug load 3.97%

10% solids Attribute Control value Example value Lot # 15-157D 15-157CHomopolymer (type/ 6.5 kDa PLA 6.5 kDa PLA amount) Copolymer (type/ 16/5PLA-PEG, 40% 16/5 PLA-PEG, 40% amount) Drug (amount DTXL) 20% 20%Organic solvent (type/ Ethyl acetate (EA), 79% Ethyl acetate (EA),amount) 79% Organic cosolvent Benzyl alcohol (BA), Benzyl alcohol (BA),(type/amount) 21% 21% Water phase 0.5% sodium cholate, 2% 0.5% sodiumcholate, BA, 4% EA in water 2% BA, 4% EA in water [solids] in oil phase5 wt % 10 wt % RESULTS Particle size 114.7 nm 115.1 nm Drug load 3.97%13.36%

20% solids Attribute Control value Example value Lot # 15-157D 15-157AHomopolymer (type/ 6.5 kDa PLA 6.5 kDa PLA amount) Copolymer (type/ 16/5PLA-PEG, 40% 16/5 PLA-PEG, 40% amount) Drug (amount DTXL) 20% 20%Organic solvent (type/ Ethyl acetate (EA), 79% Ethyl acetate (EA),amount) 79% Organic cosolvent Benzyl alcohol (BA), Benzyl alcohol (BA),(type/amount) 21% 21% Water phase 0.5% sodium cholate, 2% 0.5% sodiumcholate, BA, 4% EA in water 2% BA, 4% EA in water [solids] in oil phase5 wt % 20 wt % RESULTS Particle size 114.7 nm 130.3 nm Drug load 3.97%16.15%

40% solids Attribute Control value Example value Lot # 15-157D 15-171AHomopolymer (type/ 6.5 kDa PLA 6.5 kDa PLA amount) Copolymer (type/ 16/5PLA-PEG, 40% 16/5 PLA-PEG, 40% amount) Drug (amount DTXL) 20% 20%Organic solvent (type/ Ethyl acetate (EA), 79% Ethyl acetate (EA),amount) 79% Organic cosolvent Benzyl alcohol (BA), Benzyl alcohol (BA),(type/amount) 21% 21% Water phase 0.5% sodium cholate, 2% 0.5% sodiumcholate, BA, 4% EA in water 2% BA, 4% EA in water [solids] in oil phase5 wt % 40 wt % RESULTS Particle size 114.7 nm 130 nm Drug load 3.97%14.07%

30% solids with higher surfactant concentration for particle sizereduction; targeted nanoparticle batch. Attribute Control value Examplevalue Lot # 15-157D 35-03A Homopolymer 6.5 kDa PLA 8.2 kDa PLA(type/amount) Copolymer 16/5 PLA-PEG, 40% 16/5 PLA-PEG, 40%,(type/amount) with 1 wt % as GL2- PEG-PLA Drug (amount DTXL) 20% 20%Organic solvent Ethyl acetate (EA), Ethyl acetate (EA), (type/amount)79% 79% Organic cosolvent Benzyl alcohol (BA), Benzyl alcohol (BA),(type/amount) 21% 21% Water phase 0.5% sodium cholate, 1% sodiumcholate, 2% BA, 4% EA in 2% BA, 4% EA in water water [solids] in oilphase 5 wt % 30 wt % RESULTS Particle size 114.7 nm 114.1 nm Drug load3.97% 11.85%

Example 8 Nanoparticle Preparation—Emulsion Process 2

An organic phase is formed composed of a mixture of docetaxel (DTXL) andpolymer (homopolymer, co-polymer, and co-polymer with ligand). Theorganic phase is mixed with an aqueous phase at approximately a 1:5ratio (oil phase:aqueous phase) where the aqueous phase is composed of asurfactant and some dissolved solvent. In order to achieve high drugloading, about 30% solids in the organic phase is used.

The primary, coarse emulsion is formed by the combination of the twophases under simple mixing or through the use of a rotor statorhomogenizer. The rotor/stator yielded a homogeneous milky solution,while the stir bar produced a visibly larger coarse emulsion. It wasobserved that the stir bar method resulted in significant oil phasedroplets adhering to the side of the feed vessel, suggesting that whilethe coarse emulsion size is not a process parameter critical to quality,it should be made suitably fine in order to prevent yield loss or phaseseparation. Therefore the rotor stator is used as the standard method ofcoarse emulsion formation, although a high speed mixer may be suitableat a larger scale.

The primary emulsion is then formed into a fine emulsion through the useof a high pressure homogenizer. The size of the coarse emulsion does notsignificantly affect the particle size after successive passes (103)through the homogenizer. M-110-EH (FIG. 5).

Homogenizer feed pressure was found to have a significant impact onresultant particle size. On both the pneumatic and electric M-110EHhomogenizers, it was found that reducing the feed pressure also reducedthe particle size (FIG. 6). Therefore the standard operating pressureused for the M-110EH is 4000-5000 psi per interaction chamber, which isthe minimum processing pressure on the unit. The M-110EH also has theoption of one or two interaction chambers. It comes standard with arestrictive Y-chamber, in series with a less restrictive 200 μmZ-chamber. It was found that the particle size was actually reduced whenthe Y-chamber was removed and replaced with a blank chamber.Furthermore, removing the Y-chamber significantly increases the flowrate of emulsion during processing.

After 2-3 passes the particle size was not significantly reduced, andsuccessive passes can even cause a particle size increase. The resultsare summarized in FIG. 7, where placebo organic phase consisted of 25.5%polymer stock of 50:50 16.5/5 PLA/PEG:8.2 PLA. Organic phase wasemulsified 5:1 O:W with standard aqueous phase, and multiple discreetpasses were performed, quenching a small portion of emulsion after eachpass. The indicated scale represents the total solids of theformulation.

The effect of scale on particle size showed surprising scale dependence.The trend shows that in the 2-10 g batch size range, larger batchesproduce smaller particles. It has been demonstrated that this scaledependence is eliminated when considering greater than 10 g scalebatches. The amount of solids used in the oil phase was about 30%. FIGS.8 and 9 depicts the effect of solids concentration on particle size anddrug loading; with the exception of the 15-175 series, all batches areplacebo. For placebo batches the value for % solids represents the %solids were drug present at the standard 20% w/w.

Table A summarizes the emulsification process parameters.

TABLE A Parameter Value Observation Coarse emulsion Rotor stator Coarseemulsion size does not affect final particle size, but large formationhomogenizer coarse emulsion can cause increased oil phase retention infeed vessel Homogenizer feed 4000-5000 psi per Lower pressure reducesparticle size pressure chamber Interaction 2 × 200 μm Z- 200 μmZ-chamber yields the smallest particle size, and allows for chamber(s)chamber highest homogenizer throughput Number of 2-3 passes Studies haveshown that the particle size is not significantly reduced homogenizerpasses after 2 discreet passes, and size can even increase withsuccessive passes Water phase 0.1% [Sodium cholate] can effectivelyalter particle size; value is optimized [sodium cholate] for givenprocess and formulation W:O ratio 5:1 Lowest ratio without significantparticle size increase is ~5:1 [Solids] in oil phase 30% Increasedprocess efficiency, increased drug encapsulation, workable viscosity

The fine emulsion is then quenched by addition to deionized water at agiven temperature under mixing. In the quench unit operation, theemulsion is added to a cold aqueous quench under agitation. This servesto extract a significant portion of the oil phase solvents, effectivelyhardening the nanoparticles for downstream filtration. Chilling thequench significantly improved drug encapsulation. The quench:emulsionratio is approximately 5:1.

A solution of 35% (wt %) of Tween 80 is added to the quench to achieveapproximately 2% Tween 80 overall After the emulsion is quenched asolution of Tween-80 is added which acts as a drug solubilizer, allowingfor effective removal of unencapsulated drug during filtration. Table Bindicates each of the quench process parameters.

TABLE B Summary quench process parameters. Parameter Value ObservationInitial quench <5° C. Low temperature yields higher drug encapsulationtemperature [Tween-80] solution 35% Highest concentration that can beprepared and readily disperses in quench Tween-80:drug ratio 25:1Minimum amount of Tween-80 required to effectively remove unencapsulateddrug Q:E ratio 5:1 Minimum Q:E ratio while retaining high drugencapsulation Quench ≦5° C. (with Temperature which prevents significantdrug leaching during hold/processing temp current 5:1 Q:E quench holdtime and initial concentration step ratio, 25:1 Tween- 80:drug ratio)

The temperature must remain cold enough with a dilute enough suspension(low enough concentration of solvents) to remain below the T_(g) of theparticles. If the Q:E ratio is not high enough, then the higherconcentration of solvent plasticizes the particles and allows for drugleakage. Conversely, colder temperatures allow for high drugencapsulation at low Q:E ratios (to ˜3:1), making it possible to run theprocess more efficiently.

The nanoparticles are then isolated through a tangential flow filtrationprocess to concentrate the nanoparticle suspension and buffer exchangethe solvents, free drug, and drug solubilizer from the quench solutioninto water. A regenerated cellulose membrane is used with a molecularweight cutoffs (MWCO) of 300.

A constant volume diafiltration (DF) is performed to remove the quenchsolvents, free drug and Tween-80. To perform a constant-volume DF,buffer is added to the retentate vessel at the same rate the filtrate isremoved. The process parameters for the TFF operations are summarized inTable C. Crossflow rate refers to the rate of the solution flow throughthe feed channels and across the membrane. This flow provides the forceto sweep away molecules that can foul the membrane and restrict filtrateflow. The transmembrane pressure is the force that drives the permeablemolecules through the membrane.

TABLE C TFF Parameters Optimized Parameter Value Effect MembraneRegenerated No difference in performance between RC and PES, butMaterial cellulose - solvent compatibility is superior for RC. CoarseScreen Membrane Molecular Weight 300 kDa No difference in NPcharacteristics (i.e. residual Cut off tween)Increase in flux rates isseen with 500 kDa membrane but 500 kDa is not available in RC CrossflowRate 11 L/min/m² Higher crossflow rate led to higher flux Transmembrane20 psid Open channel membranes have maximum flux rates Pressure between10 and 30 psid. Coarse channel membranes have maximum flux rates withmin TMP (~20 psid). Concentration of 30 mg/ml Diafiltration is mostefficient at [NP] ~50 mg/ml with Nanoparticle open channel TFF membranesbased on flux rates and Suspension for throughput. With coarse channelmembranes the flux rate Diafiltration is optimized at ~30 mg/ml in thestarting buffer. Number of ≧15 (based on About 15 diavolumes are neededto effectively remove Diavolumes flux increase) tween-80. End point ofdiafiltration is determined by in- process control (flux increaseplateau). Membrane Area ~1 m²/kg Membranes sized based on anticipatedflux rates and volumes required.

The filtered nanoparticle slurry is then thermal cycled to an elevatedtemperature during workup. A small portion (typically 5-10%) of theencapsulated drug is released from the nanoparticles very quickly afterits first exposure to 25° C. Because of this phenomenon, batches thatare held cold during the entire workup are susceptible to free drug ordrug crystals forming during delivery or any portion of unfrozenstorage. By exposing the nanoparticle slurry to elevated temperatureduring workup, this ‘loosely encapsulated’ drug can be removed andimprove the product stability at the expense of a small drop in drugloading. Table D summarizes two examples of 25° C. processing. Otherexperiments have shown that the product is stable enough after ˜2-4diavolumes to expose it to 25° C. without losing the majority of theencapsulated drug. 5 diavolumes is used as the amount for coldprocessing prior to the 25° C. treatment.

TABLE D Lots A Lots B Drug load Cold workup 11.3% 9.7% 25° C. workup¹8.7-9.1% 9.0-9.9% Stability² Cold workup <1 day <1 day 25° C. workup¹5-7 days  2-7 days In vitro burst³ Cold workup  ~10% Not 25° C. workup¹  ~2% performed ¹25° C. workup sublots were exposed to 25° C. after atleast 5 diavolumes for various periods of time. Ranges are reportedbecause there were multiple sublots with 25° C. exposure. ²Stabilitydata represents the time that final product could be held at 25° C. at10-50 mg/ml nanoparticle concentrations prior to crystals forming in theslurry (visible by microscopy) ³In vitro burst represents the drugreleased at the first time point (essentially immediately)

After the filtration process the nanoparticle suspension is passedthrough a sterilizing grade filter (0.2 μm absolute). Pre-filters areused to protect the sterilizing grade filter in order to use areasonable filtration area/time for the process. Values are assummarized in Table E.

TABLE E Parameter O Value Effect Nanoparticle   50 mg/ml Yield lossesare higher at higher [NP], Suspension but the ability to filter at 50mg/ml Concentration obviates the need to aseptically concentrate afterfiltration Filtration flow ~1.3 L/min/m² Filterability decreases as flowrate rate increases

The filtration train is Ertel Alsop Micromedia XL depth filter M953Pmembrane (0.2 μm Nominal); Pall SUPRAcap with Seitz EKSP depth filtermedia (0.1-0.3 μm Nominal); Pall Life Sciences Supor EKV 0.65/0.2 micronsterilizing grade PES filter.

0.2 m² of filtration surface area per kg of nanoparticles for depthfilters and 1.3 m2 of filtration surface area per kg of nanoparticlesfor the sterilizing grade filters can be used.

Example 9

Target-specific nanoparticles can be prepared that include abiocompatible polymer congutated to e.g. PEG, the chemotherapeuticsdescribed herein, and optionally conjugated to GL1 or GL2. Exemplarynanoparticles are shown in Table 1 below:

TABLE 1 Nanoparticles having a listed therapeutic agent and a polymerconjugate comprising: Biocompatible Polymer-Polymer -(Targeting Moiety)Targeting Biocompatible Moiety Therapeutic Agent Polymer Polymer(Optional) vincristine PLGA PEG GL1 vincristine PLA PEG GL1 vincristinePGA PEG GL1 vincristine PLGA PEG GL2 vincristine PLA PEG GL2 vincristinePGA PEG GL2 vincristine PLGA PEG-DSPE GL1 vincristine PLA PEG-DSPE GL1vincristine PGA PEG-DSPE GL1 vincristine PLGA PEG-DSPE GL2 vincristinePLA PEG-DSPE GL2 vincristine PGA PEG-DSPE GL2 docetaxel PLGA PEG GL1docetaxel PLA PEG GL1 docetaxel PGA PEG GL1 docetaxel PLGA PEG GL2docetaxel PLA PEG GL2 docetaxel PGA PEG GL2 docetaxel PLGA PEG-DSPE GL1docetaxel PLA PEG-DSPE GL1 docetaxel PGA PEG-DSPE GL1 docetaxel PLGAPEG-DSPE GL2 docetaxel PLA PEG-DSPE GL2 docetaxel PGA PEG-DSPE GL2sirolimus PLGA PEG GL1 sirolimus PLA PEG GL1 sirolimus PGA PEG GL1sirolimus PLGA PEG GL2 sirolimus PLA PEG GL2 sirolimus PGA PEG GL2sirolimus PLGA PEG-DSPE GL1 sirolimus PLA PEG-DSPE GL1 sirolimus PGAPEG-DSPE GL1 sirolimus PLGA PEG-DSPE GL2 sirolimus PLA PEG-DSPE GL2sirolimus PGA PEG-DSPE GL2 gemcitabine PLGA PEG GL1 gemcitabine PLA PEGGL1 gemcitabine PGA PEG GL1 gemcitabine PLGA PEG GL2 gemcitabine PLA PEGGL2 gemcitabine PGA PEG GL2 gemcitabine PLGA PEG-DSPE GL1 gemcitabinePLA PEG-DSPE GL1 gemcitabine PGA PEG-DSPE GL1 gemcitabine PLGA PEG-DSPEGL2 gemcitabine PLA PEG-DSPE GL2 gemcitabine PGA PEG-DSPE GL25-fluorouracil PLGA PEG GL1 5-fluorouracil PLA PEG GL1 5-fluorouracilPGA PEG GL1 5-fluorouracil PLGA PEG GL2 5-fluorouracil PLA PEG GL25-fluorouracil PGA PEG GL2 5-fluorouracil PLGA PEG-DSPE GL15-fluorouracil PLA PEG-DSPE GL1 5-fluorouracil PGA PEG-DSPE GL15-fluorouracil PLGA PEG-DSPE GL2 5-fluorouracil PLA PEG-DSPE GL25-fluorouracil PGA PEG-DSPE GL2 paclitaxel PLGA PEG GL1 paclitaxel PLAPEG GL1 paclitaxel PGA PEG GL1 paclitaxel PLGA PEG GL2 paclitaxel PLAPEG GL2 paclitaxel PGA PEG GL2 paclitaxel PLGA PEG-DSPE GL1 paclitaxelPLA PEG-DSPE GL1 paclitaxel PGA PEG-DSPE GL1 paclitaxel PLGA PEG-DSPEGL2 paclitaxel PLA PEG-DSPE GL2 paclitaxel PGA PEG-DSPE GL2 daunorubicinPLGA PEG GL1 daunorubicin PLA PEG GL1 daunorubicin PGA PEG GL1daunorubicin PLGA PEG GL2 daunorubicin PLA PEG GL2 daunorubicin PGA PEGGL2 daunorubicin PLGA PEG-DSPE GL1 daunorubicin PLA PEG-DSPE GL1daunorubicin PGA PEG-DSPE GL1 daunorubicin PLGA PEG-DSPE GL2daunorubicin PLA PEG-DSPE GL2 daunorubicin PGA PEG-DSPE GL2

Example 10

Nanoparticles shown in Table 2 are prepared using the procedure inExample 8. Nanoparticles comprising macromolecules of PLGA-PEG andmacromolecules of PLGA-PEG-small-molecule ligand (SML) were prepared asshown in studies 1 and 2, below. In studies 3 and 4, nanoparticlescomprising macromolecules of PLA-PEG, macromolecules of PLGA-PEG-SML,and macromolecules of PLA were prepared (DB=diblock copolymer).

The ratio of small-molecule targeting moiety-functionalizedmacromolecules with nonfunctionalized macromolecules can be adjusted,and using study 1, nanoparticles with polymer compositions that areapproximately 0.94 mole %, 4.63 mole % and 9.01 mole % functionalizedmacromolecules can be prepared (see “mol % DB-GL2 of total Poly”).Additionally, using these methods, nanoparticles comprisingapproximately 0.015, 0.073 and 0.143 weight % small-molecule ligand withrespect to total polymer can be prepared (see “Wt. % GL2 wrt poly.”).

Nanoparticles with functionalized polymers that constitute approximately0.1-30, e.g., 0.1-20, e.g., 0.1-10 mole percent of the entire polymercomposition of the nanoparticle can also be prepared, as well asnanoparticles having a weight percent low-molecular weight ligand withrespect to total polymer is between 0.001 and 5, e.g., 0.001 and 2,e.g., 0.001 and 1.

TABLE 2 Wt. % of Wt. % of wt. % DB-GL2 mol % DB-GL2 Solids Polymer ofPLA-PEG of total Poly. Study 1 0.362 0.381052632 NA 0.947217483 1.811.905263158 NA 4.630814102 3.62 3.810526316 NA 9.011251618 Study 2 0.1810.190526316 NA 0.474958408 0.362 0.381052632 NA 0.947217483 0.5430.571578947 NA 1.416800171 1.81 1.905263158 NA 4.630814102 Study 3 0.3620.4525 NA 0.178974269 1.81 2.2625 NA 0.891043972 Study 4 0.0802410.100300903 0.200601805 0.079390136 Calc. for 0.161616 0.2020202020.404040404 0.159825753 45K-5K PLA-PEG 0.842105 1.052631579 2.1052631580.829427309 1.702128 2.127659574 4.255319149 1.668024361 Study 40.190522 0.238151941 0.476303882 0.16998719 Calc. for 16K-5K PLA-PEG0.381134 0.476417342 0.952834683 0.340027827 1.909308 2.3866348454.77326969 1.702280075 3.827751 4.784688995 9.56937799 3.409927685 NOPLA 0.323232 0.404040404 0.404040404 0.159825753 Wt. % GL2 GL2 contentMol. % GL2 wrt poly. ppm Study 1 0.947217483 0.015108 151.08124.630814102 0.073861 738.6148 9.011251618 0.143729 1437.295 Study 20.474958408 0.007576 75.75587 0.947217483 0.015108 151.0812 1.4168001710.022598 225.9796 4.630814102 0.073861 738.6148 Study 3 0.1789742690.002855 28.5464 0.891043972 0.014212 142.1215 Study 4 0.0793901360.001266 12.66273 Calc. for 45K-5K PLA-PEG 0.159825753 0.002549 25.492210.829427309 0.013229 132.2937 1.668024361 0.026605 266.0499 Study 40.16998719 0.002711 27.11296 Calc. for 16K-5K PLA-PEG 0.3400278270.005423 54.23444 1.702280075 0.027151 271.5137 3.409927685 0.054388543.8835 NO PLA 0.159825753 0.002549 25.49221

Example 11

Various nanoparticle formulations are formed using the procedure ofExample 8 as depicted and compared in Table F:

TABLE F % Formulation Polymer Type Solids Load and particle sizePolymer-PEG:PLA Ratio (80:0; 16-5 PLA-PEG:PLA  5% 60:20; 40:40(baseline), 20:60) Ratio 45-5 PLGA-PEG:PLA 5% Ratio PLA Molecular Weight= 1.9, 4, 6.5 16-5 PLA-  5% 1.9 and 4 kDa had lower load = 2.5%(baseline), 8.5 kDa PEG:PLA (40:40) 15-5 Vs 16-5 PLA-PEG:PLA (40:40)15-5 PLA-  5% Both 15-5 PLA-PEG and 16-5 PLA-PEG PEG:PLA are the same inload and particle size (40:40) Total % Solid 5% Vs. 15% 16-5 PLA- 5% or15% When use 15% solids; 3X greater PEG:PLA encapsulation efficiency(40:40) 16-5 PLGA-PEG Vs. PLA-PEG 16-5 PLGA- 15% Both 16-5 PLGA-PEG and16-5 PLA- (baseline) with PLA (40:40) PEG:PLA PEG are equivalent as to %load and (40:40) particle size Alternative polymer: PLGA-PEG 28-5 PLGA-15% 28-5 PLGA-PEG = larger particle size as PEG:PLA compared to others(40:40) 45-5 PLGA- 15% 45-5 PLGA-PEG = larger particle size PEG:PLA(40:40) Ratio of Benzyl Alcohol to Ethyl 16-5 PLA- 15% Ratio = 21:79(10.8% load); 32:68 and Acetate: 11:89, 21:79 (baseline), 32:68 PEG:PLA11:89 resulted in 9.4 and 8.8% load, BA:EA (40:40) respectively. CompareSolvent to Benzyl Alcohol: 16-5 PLA- 15% Solvent = benzyl alcohol (10.8%load); Heptanol or Hexanol PEG:PLA heptanol and hexanol both resulted in~2% (40:40) load Target Load 10, 20 (baseline), 30% 16-5 PLA- 15% Loadincreased with target load: % Load = PEG:PLA 5.8%, 9%, 13.3%,respectively (40:40)

An optimal particle size can be achieved without using homopolymer PLAand without significantly sacrificing drug load, as shown in FIG. 10.Batches with PLA homopolymer release drug significantly faster thanbatches made using co-polymer alone FIG. 11). The various polymer typesand molecular weights added no additional value in optimizing drug loadand particle size. To the contrary, at 15% total solids with“alternative polymer” types particle size were typically larger than atarget size of 100-120 nm. Cetyl alcohol at 5 wt % incorporationgenerally increased the rate of in vitro release (FIG. 12).

Example 12 Cryoprotectant

Freezing a suspension of nanoemulsion nanoparticles in deionized wateralone results in particle aggregation. This is believed to be due tocrystallization and entanglement of PEG chains on the nanoparticlesurfaces (Jaeghere et al; Pharmaceutical Research 16(6), p 859-852).Sugar-based excipients (sucrose, trehalose, or mannitol) can act tocryoprotect these nanoparticles under freeze/thaw conditions, with aconcentrations as low as 1 wt % for dilute (˜10 mg/ml) nanoparticlesuspensions. One formulation includes 10 wt % sucrose, which containsexcess sucrose to what is required and is the same osmolality asphysiological saline.

Table G shows that 16/5 PLA-PEG co-polymer is less susceptible tofreeze-thaw aggregation.

TABLE G Original Post-F/T Post-F/T Median PSD/ Median PS Poly- Post-F/TDescription PD (nm) dispersity Baseline Index 1:1 45/5 and PLA 143.4,0.124 358.9 0.358 0.0/23.16% (baseline) 16/5 PLA-PEG and PLA 186.7,0.080 189.5 0.126 9.7/91.57% (1:1) 2:1:1 16/5:PLA:cetyl 174.1, 0.084232.7 0.146 0.0/61.19% 2:1:1 45/5:PLA:cetyl 111.0, 0.182 0 0 0.0/1.55% 16/5 PLA-PEG alone 218.8, 0.098 226.9 0.03 7.3/60.56% 16/5 PLA-PEG andPLA 222.2, 0.126 230.7 0.065 4.1/35.36% (3:1) 45/5 PLGA-PEG and 162.7,0.099 178.6 0.091 7.7/95.41% PLA (3:1) 2:1:1 45/5 PLA- 115.9, 0.154734.6 0.392 0.0/13.27% PEG:PLA:cetyl

Example 13 Palladium Removal

Based on a dose level (ug/day) in a human clinical trials, a maximumacceptable palladium level in a PLA-PEG-GL2 composition is ca. 10 ppm.Polymer (PLA-PEG-GL2) solutions (20 or 35 mg/mL) in dichloromethane(DCM) were loaded on to 5 g resin columns (pre-solvated with 10 mL DCM)and subsequent eluted using 30 mL DCM under gravity. Polymer wasrecovered by solvent removal using rotary evaporation followed by vacuumdrying at room temperature. Polymer recovery was determinedgravimetrically and residual palladium content was determined byInductively Coupled Plasma (ICP) Spectroscopy at Galbraith LaboratoriesInc.

TABLE H Resin PLA-PEG-GL2 solution and yield Palladium content (ppm)used Solvent mg/mL mg/5 g resin wt. % recovery Test 1 Test 2 AverageGuanidine DCM 20 220 23 337 347 342 Thiol DCM 20 220 62 39 30 34.5 TMTDCM 20 220 92 11 7 9 Urea DCM 20 220 60 4470 NA 4470 Thiourea DCM 20 22045 40 36 38 Control DCM 20 NA NA 4060 3980 4020 TMT DCM 35 335 91 9 7 8Urea DCM 35 335 60 5360 4920 5140 Control DCM NA NA NA 4240 4300 4270TMT DCM 35 1050  92 3.8 2.7 3.25 Control DCM NA NA NA 3780 3880 3830

As seen in Table H, thiol, TMT, urea and thiourea functionalitiesbrought palladium levels below 50 ppm at the polymer load per unit resinweight evaluated. However, only the TMT (trimercaptotriazine) resinyielded good (>90%) polymer recovery. In addition, TMT resin alsoyielded palladium contents under the 10 ppm acceptance threshold. Thereappear to be some variability in the results depending on experimentalconditions used. In particular, palladium removal is more effective whenthe 5 g TMT resin column was loaded with 1050 mg polymer. This may bedue to longer residence time of the polymeric species and palladiumcatalyst under these experimental conditions.

Example 14 Formulation

A formulation that includes nanoparticles of PLA-PEG-ligand, PLA,PLA-PEG, and docetaxel, in a sucrose/water composition is formed:

Component Nominal Concentration (mg/mL) Docetaxel 5 PLA-PEG-ligand 1.1PLA-PEG 21.4 PLA 22.5 Sucrose 100 Water Q.S.

Example 15 In Vitro Release

An in vitro release method is used to determine the initial burst phaserelease from these nanoparticles at both ambient and 37° C. conditions.In order to maintain sink conditions and prevent nanoparticles fromentering the release samples, a dialysis system was designed. Afterobtaining an ultracentrifuge capable of pelleting 100 nm particles, thedialysis membranes were eliminated and centrifugation was used toseparate released drug from encapsulated drug.

The dialysis system is as follows: 3 mL slurry of docetaxelnanoparticles (approx 250 μg/mL drug/PLGA/PLA nanoparticles,corresponding to 2.5 mg/mL solid concentration) in DI-water is placedinto the inner tube of a 300 kDa MWCO dialyzer by pipetting. Thenanoparticle is suspension in this media. The dialyzer is placed into aglass bottles containing 130 ml release media (2.5% hydroxyl betacyclodextrin in PBS), which is continually stirred at 150 rpm using ashaker to prevent the formation of an unstirred water layer at themembrane/outer solution interface. At pre-determined time points,aliquot of samples (1 mL) were withdrawn from the outer solution(dialysate) and analyzed for Docetaxel concentration by HPLC.

The centrifugal system is run using similar conditions at lowersuspension volumes without dialysis bags. Samples are centrifuged at60,000 g for 30 minutes and the supernatant is assayed for drug contentto measured released drug.

Example 16 In Vitro Release of Docetaxel Nanoparticles

A suspension of docetaxel nanoparticles prepared as in Example 8 (10% byweight docetaxel and 90% by weight polymer (1.25 wt % PLA-PEG-GL2 and98.75 wt % PLA-PEG, Mn PLA=16 Da; Mn PEG=5 Da) were placed in a dialysiscassette and incubated in a reservoir of PBS at 37 C with stirring.Sample of the dialysate were collected and analyzed for docetaxel usingreversed phase HPLC. For comparison, conventional docetaxel wassubjected to the same procedure. FIG. 13 depicts in vitro releaseprofile of nanoparticles compared to the conventional docetaxel. Releaseof the encapsulated docetaxel from the polymer matrix was essentiallylinear over the first 24 hours with the remainder gradually releasedfrom the particles over a period of about 96 hours.

Example 17 Sirolimus Nanoparticles

Nanoparticle batches were prepared using the general procedure ofExample 8, with 80% (w/w) Polymer-PEG or Polymer-PEG with homopolymerPLA at 40% (w/w) each, with a batch of % total solids of 5%, 15% and30%. Solvents used were: 21% benzyl alcohol and 79% ethyl acetate (w/w).For each 2 gram batch size, 400 mg of drug was used and 1.6 g of 16-5Polymer-PEG or 0.8 g of 16-5 Polymer-PEG+0.8 g of 10 kDa PLA(homopolymer) was used. The diblock polymer 16-5 PLA-PEG or PLGA-PEG(50:50 L:G) was used, and if used, the homopolymer: PLA with a Mn=6.5kDa, Mw=10 kDa, and Mw/Mn=1.55.

The organic phase (drug and polymer) is prepared in 2 g batches: To 20mL scintillation vial add drug and polymer(s). The mass of solventsneeded at % solids concentration is shown below:

i. 5% solids: 7.98 g benzyl alcohol+30.02 g ethyl acetate

ii. 15% solids: 2.38 g benzyl alcohol+8.95 g ethyl acetate

iii. 30% solids: 0.98 g benzyl alcohol+3.69 g ethyl acetate

An aqueous solution is prepared with 0.5% sodium cholate, 2% benzylalcohol, and 4% ethyl acetate in water. To a 2 L bottle add 7.5 g sodiumcholate, 1402.5 g of DI water, 30 g of benzyl alcohol and 60 g of ethylacetate, and mix on stir plate until dissolved.

For the formation of emulsion, a ratio of aqueous phase to oil phase of5:1 is used. The organic phase is poured into the aqueous solution andhomogenized using IKA for 10 seconds at room temperature to form courseemulsion. The solution is fed through the homogenizer (110S) at 9 Kpsi(45 psi on gauge) for 2 discreet passes to form nanoemulsion.

The emulsion is poured into quench (D.I. water) at <5 C while stirringon stir plate. Ratio of quench to emulsion is 8:1.35% (w/w) Tween 80 isadded in water to quench at ratio of 25:1 Tween 80 to drug. Thenanoparticles are concentrated through TFF and the quench isconcentrated on TFF with 500 kDa Pall cassette (2 membrane) to ˜100 mL.Diafiltering is used using ˜20 diavolumes (2 liter) of cold DI water,and the volume is brought down to minimal volume then collect finalslurry, ˜100 mL. The solids concentration of unfiltered final slurry isdetermined by the using tared 20 mL scintillation vial and adding 4 mLfinal slurry and dry under vacuum on lyo/oven and the weight ofnanoparticles in the 4 mL of slurry dried down is determined.Concentrated sucrose (0.666 g/g) is added to final slurry sample toattain 10% sucrose.

Solids concentration of 0.45 um filtered final slurry was determined byfiltering about 5 mL of final slurry sample before addition of sucrosethrough 0.45 μm syringe filter; to tared 20 mL scintillation vial add 4mL of filtered sample and dry under vacuum on lyo/oven.

The remaining sample of unfiltered final slurry was frozen with sucrose.Rapamycin (sirolimus) formulations:

Drug Release of Drug (t = hr) Name Polymer Size (nm) Loading T = 0 T = 2T = 4 T = 24  5% Solid 16/5 PLA/PEG 123.1  3.61% ND ND ND ND 16/5PLA/PEG + PLA 119.7  4.49% ND ND ND ND 15% Solid 16/5 PLA/PEG 82.1 4.40% ND ND ND ND 16/5 PLA/PEG + PLA 120.6 11.51% ND ND ND ND 23% Solid16/5 PLA/PEG 88.1  7.40% ND ND ND ND 16/5 PLA/PEG + PLA 118.3  7.8% NDND ND ND 30% Solid 16/5 PLA/PEG 88.5 10.26% 8.5 17.3 22.4 64.2 16/5PLA/PEG + PLA 118.3 10.18% 9.3 30.4 44.7 98.2

The effect of solid contents and the inclusions of poly(lactic) acidhomopolymer is shown in FIG. 14.

In-vitro release experiments are studied by dispersing nanoparticles inPBS containing 10% (w/w) of Tween 20 (T20) at 37° C. T20 was used toincrease the solubility of rapamycin in PBS to levels well detectable byHPLC as well as maintaining the sink condition. 3 mL of drug-loadednanoparticles were redispersed in 130 mL of release medium in a jar at aknown concentration (approximately 250 μg/ml). These volumes were chosento ensure that the maximum concentration of the drug in the releasemedium would always be less than 10% of the maximum solubility, i.e.,sink conditions. The media and nanoparticle suspension is stirred at 150rpm. At pre-determined time points, 4 ml of aliquots were centrifuged at50,000 rpm (236,000 g) for 1 hr to separate the nanoparticles from theelution media. The elution media is injected in to a HPLC to determinedrug released from the nanoparticles. The release of rapamycin showedslow and sustained release, as shown in FIG. 15.

Example 18 Temsirolimus

Nanoparticles were prepared as in Example 17 and 8, except temsirolimuswas used with 30% solid content in the organic phase before emulsion:

Size Drug Release of Drug (t = hr) Name Polymer (nm) Loading T = 0 T = 2T = 4 T = 24 30% 16/5 PLA/PEG 97.5 9.9% 11.5 15.6 17.9 40.9 Solid 16/5PLA/PEG + 112.8 14.2% 9.8 22.3 29.9 88.0 PLA 16/5 PLGA/ 150.3 4.6 ND NDND ND PEG + PLA 16/5 PLGA/ ND 6.9 10.6 35.7 45.8 87.0 PEG + PLA

FIG. 16 depicts the weight % of temsirolimus and FIG. 17 depicts thenanoparticle for the different polymeric nanoparticles havingtemsirolimus. The results of an in-vitro release experiment as inExample 17 shows the slow and sustained release of temsirolimus showedslow and sustained release, as shown in FIG. 18.

Example 19 Vinorelbine Nanoparticles

Nanoparticle batches were prepared using the general procedure ofExample 8, with 80% (w/w) Polymer-PEG or Polymer-PEG with homopolymerPLA at 40% (w/w) each, with a batch of % total solids of 5%, 15% and30%. Solvents used were: 21% benzyl alcohol and 79% ethyl acetate (w/w).For each 2 gram batch size, 400 mg of vinorelbine was used and 1.6 g of16-5 Polymer-PEG or 0.8 g of 16-5 Polymer-PEG+0.8 g of 10 kDa PLA(homopolymer) was used. The diblock polymer 16-5 PLA-PEG or PLGA-PEG(50:50 L:G) was used, and if used, the homopolymer: PLA with a Mn=6.5kDa, Mw=10 kDa, and Mw/Mn=1.55.

The organic phase (drug and polymer) is prepared in 2 g batches: To 20mL scintillation vial add drug and polymer(s). The mass of solventsneeded at % solids concentration is shown below:

i. 5% solids: 7.98 g benzyl alcohol+30.02 g ethyl acetate

ii. 15% solids: 2.38 g benzyl alcohol+8.95 g ethyl acetate

iii. 30% solids: 0.98 g benzyl alcohol+3.69 g ethyl acetate

An aqueous solution is prepared with 0.5% sodium cholate, 2% benzylalcohol, and 4% ethyl acetate in water. Add to the bottle 7.5 g sodiumcholate, 1402.5 g of DI water, 30 g of benzyl alcohol and 60 g of ethylacetate, and mix on stir plate until dissolved.

For the formation of emulsion, a ratio of aqueous phase to oil phase is5:1. The organic phase is poured into the aqueous solution andhomogenized using IKA for 10 seconds at room temperature to form courseemulsion. The solution is fed through the homogenizer (110S) at 9 Kpsi(45 psi on gauge) for 2 discreet passes to form nanoemulsion.

The emulsion is poured into quench (D.I. water) at <5° C. while stirringon stir plate. Ratio of quench to emulsion is 8:1.35% (w/w) Tween 80 isadded in water to quench at ratio of 25:1 Tween 80 to drug. Thenanoparticles are concentrated through TFF and the quench isconcentrated on TFF with 500 kDa Pall cassette (2 membrane) to ˜100 mL.Diafiltering is used using ˜20 diavolumes (2 liters) of cold DI water,and the volume is brought down to minimal volume then collect finalslurry, ˜100 mL. The solids concentration of unfiltered final slurry isdetermined by the using tared 20 mL scintillation vial and adding 4 mLfinal slurry and dry under vacuum on lyo/oven and the weight ofnanoparticles in the 4 mL of slurry dried down is determined.Concentrated sucrose (0.666 g/g) is added to final slurry sample toattain 10% sucrose.

Solids concentration of 0.45 um filtered final slurry was determined byfiltering about 5 mL of final slurry sample before addition of sucrosethrough 0.45 μm syringe filter; to tared 20 mL scintillation vial add 4mL of filtered sample and dry under vacuum on lyo/oven.

The remaining sample of unfiltered final slurry was frozen with sucrose.Vinorelbine Formulations:

In-vitro Particle % release % Vinorelbine Size Solids conducted PolymerType: Load (HPLC) (nm)  5% 16-5 PLA-PEG 4.27 143.3 16-5 PLA-PEG + PLA3.39 105.7 15% 16-5 PLA-PEG 6.2 100.3 16-5 PLA-PEG + PLA 15.95 141.3 30%16-5 PLA-PEG (n = 3) 10.41 90.8 10.31 84.4 * 12.01 95 * 16-5 PLA-PEG +PLA 15.03 125.5 * 16-5 PLGA-PEG + PLA 14.66 120.3 * = in-vitro releasedone on samples

In-vitro release was conducted on three formulations at 30% totalssolids: 16-5 PLA-PEG; 16-5 PLA-PEG+PLA; and 16-5 PLGA-PEG+PLA, and thein-vitro release data was collected at 37° C. in an air chamber using10% urea in PBS solution as the release media. The table below and FIG.19 depicts the results:

16-5 PLA- 16-5 PLA-PEG + 16-5 PLGA-PEG + Time Point (hours) PEG 10 kDaPLA 10 kDa PLA 0 5.62 0.84 4.79 2 35.29 35.35 67.63 5 41.28 49.58 87.0524 65.20 91.81 101.62 48 73.02 88.63 89.57 144 81.08 84.98 91.46

Example 20 Vincristine

Nanoparticle formulations that include vincristine were prepared usingthe general procedure of Example 8.

Vincristine Formulations:

Composition by Ref. No. Components Wt. (%) 50-103-3-5mPEG(5k)-lPLA(16K)/Vincristine 96/4 50-117-1-5mPEG(5k)-lPLA(16K)/Vincristine 95/5 50-117-2-5mPEG(5k)-lPLA(16K)/Vincristine 96/4 50-103-4mPEG(5k)-lPLA(16K)/lPLA(16K)/ 46/46/8 Vincristine 50-103-2mPEG(5k)-lPLA(16K)/lPLA(16K)/ 47/47/6 VincristineAnalytical Characterization of Vincristine Formulations:

Ref. No. Size (nm) Drug Load (%) Encapsulation Efficiency (%) 50-103-3-5103 4.4 21.8 50-117-1-5 110 4.6 22.8 50-117-2-5 115 4.2 20.8 50-103-4146 8.3 41.6 50-103-2 98 6.0 30.0

In vitro release was conducted on the vincristine formulations, and thein vitro release data was collected at 37° C. in an air chamber using10% urea in PBS solution as the release media. FIG. 20 depicts in-vitrorelease for several of the lots referenced.

Example 21 Pharmacokinetics

The pharmacokinetics (PK) of nanoparticles having vincristine asprepared in Example 20 and having docetaxel as prepared in Example 8were determined in Sprague-Dawley (SD) rats. Rats (male Sprague Dawley,approximately 300 g with jugular cannulae) were given a singleintravenous dose of 0.5 mg/kg free drug or passively targetednanoparticles encapsulating drug (10 wt % drug, 90 wt polymer (PLA-PEG,Mn PLA=16 Da; Mn PEG=5 Da, PTNP) with 5 mg/kg drug and PTNP at time=0.At various times after dosing, blood samples were collected from thejugular cannulae into tubes containing lithium heparin, and plasma wasprepared. Plasma levels were determined by extraction of the drugs fromplasma followed by LCMS analysis.

FIG. 21 depicts the PK profiles of vincristine and vincristine PTNP, anddocetaxel and docetaxel PTNP.

Example 22 Particle Size Analysis

Particle size is analyzed by two techniques—dynamic light scattering(DLS) and laser diffraction. DLS is performed using a BrookhavenZetaPals instrument at 25° C. in dilute aqueous suspension using a 660nm laser scattered at 90° and analyzed using the Cumulants and NNLSmethods (TP008). Laser diffraction is performed with a Horiba LS950instrument in dilute aqueous suspension using both a HeNe laser at 633nm and an LED at 405 nm, scattered at 90° and analyzed using the Mieoptical model (TP009). The output from the DLS is associated with thehydrodynamic radius of the particles, which includes the PEG ‘corona’,while the laser diffraction instrument is more closely associated withthe geometric size of the PLA particle ‘core’.

Example 23 Ligand Density

Assuming an overall particle diameter is equivalent to the hydrodynamicdiameter as measured by the Brookhaven particle sizer, nanoparticles areperfect spheres, and all of the hydrophilic PEG and ligand is expressedon the surface as well as that all of the PEG is fully hydrated, a modelof the particle surface can be built, as shown in Table I:

TABLE I Nanoparticle surface model for 100 nm particles of 16/5co-polymer and 6.5 kDa homopolymer Polymer (molecules Ligand % orSA/particle) Ligand Coverage mol % of co- Homo- Molecules/ polymerpolymer Co-polymer nm²/PEG mol/g NP particle nm²/ligand  0% (NTNP) 70502182 14.40 0 0 NA  1% GL2 7049 2183 14.39 1.72 × 10⁻⁰⁷ 22 1439   5% GL27047 2187 14.37 8.63 × 10⁻⁰⁷ 109 287 10% GL2 7043 2191 14.34 1.73 ×10⁻⁰⁷ 219 143

Example 24 Breast Cancer Tumor Targeting

The ability of intravenously administered nanoparticles prepared as inExample 8 (10 wt % docetaxel, 90 wt polymer (˜1.25 wt % PLA-PEG-GL2; and˜98.75% PLA-PEG, Mn PLA=16 Da; Mn PEG=5 Da) (labeled as B1ND-14) toinhibit non-prostate tumor growth was assessed in comparison toconventional docetaxel and non-targeted control particles having samedrug/polymer composition (PTNP) in mice implanted with MX-1 xenografts.When tumors reached an average volume of 300 mm³, mice were administeredtest articles (sucrose, docetaxel, PTNP, BIND-14) every 4 days for 3doses. Average tumor volumes over time for each treatment group is shownin FIG. 22.

The ability of targeted nanoparticles (BIND-14) to enhance the deliveryof docetaxel to tumors after intravenous administration was assessed inmice bearing human MX-1 breast cancer xenografts, with an average tumorvolume of 1700 mm³. The docetaxel concentrations (ng/mg) in tumorsexcised 24 hours after the IV dose from animals dosed with BIND-14,PTNP, and conventional docetaxel were analyzed for docetaxel contentusing LC/MS/MS and are presented in FIG. 23.

Example 24 Prostate Cancer Tumor Targeting

Nanoparticle delivery of docetaxel using nanoparticles prepared as inExample 8 (10 wt % docetaxel, 90 wt polymer (˜1.25 wt % PLA-PEG-GL2; and˜98.75% PLA-PEG, Mn PLA=16 Da; Mn PEG=5 Da; BIND-14) to tumors afterintravenous administration was assessed in male SCID mice bearing humanLNCaP prostate cancer xenografts. Male SCID mice were subcutaneouslyinoculated with human LNCaP prostate cancer cells. Three to four weeksafter inoculation, a single IV dose of 5 mg/kg docetaxel wasadministered as either BIND-014 or conventional docetaxel. Mice weresacrificed 2 h or 12 h post-dose. The tumors from each group wereexcised and assayed for docetaxel by an LC-MS method.

Twelve hours after a single dose of 50 mg/kg of BIND-14, tumordocetaxelconcentration in animals receiving BIND-014 was approximately7-times higher than in animals receiving conventional DTXL, indicatingthat long-circulating PSMA-targeted nanoparticles deliver more DTXL tothe tumor site as shown in FIG. 24.

The ability of repeat doses of BIND-014 to suppress tumor growth wasalso assessed in the LNCaP xenograft tumor model as shown in FIG. 25.Male SCID mice were subcutaneously inoculated with human LNCaP prostatecancer cells. Three to four weeks after inoculation, the mice weretreated every other day for four doses with BIND-014, conventionaldocetaxel (DTXL), DTXL encapsulated in non-targeted nanoparticles(PTNP), and vehicle (Control). After four doses of 5 mg/kg, tumor volumereduction was greater in animals receiving BIND-014 compared toconventional docetaxel or non-targeted particles (PTNP). The increase intumor docetaxel concentration results in a more pronounced cytotoxiceffect.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications,websites, and other references cited herein are hereby expresslyincorporated herein in their entireties by reference.

In one aspect, the invention provides therapeutic nanoparticle thatincludes an active agent or therapeutic agent, e.g. taxane, and one,two, or three biocompatible polymers. For example, disclosed herein is atherapeutic nanoparticle comprising about 0.2 to about 35 weight percentof a therapeutic agent; about 10 to about 99 weight percent poly(lactic)acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly (glycolic)acid-poly(ethylene)glycol copolymer; and about 0 to about 50 weightpercent poly(lactic) acid or poly(lactic) acid-co-poly (glycolic) acid.Exemplary therapeutic agents include antineoplastic agents such astaxanes, e.g. docetaxel and may include about 10 to about 30 weightpercent of a therapeutic agent, e.g., a taxane agent.

Provided herein, in part, is a method of preparing a plurality ofdisclosed therapeutic nanoparticles, comprising combining a therapeuticagent, a first polymer, and a second polymer, with an organic solvent(for example, a solvent chosen from: ethyl acetate, benzyl alcohol,methylene chloride, dimethyl formamide, Tween 80 and Span 80, andcombinations of two or more thereof) to form a first organic phasehaving about 5 to about 50% solids; combining the first organic phasewith a first aqueous solution (which may, in some embodiments, include areagent chosen from: sodium cholate, ethyl acetate, benzyl alcohol orcombinations thereof) to form a second phase; emulsifying the secondphase to form an emulsion phase; quenching the emulsion phase to form aquenched phase; adding a drug solubilizer to the quenched phase to forma solubilized phase of unencapsulated therapeutic agent; and filteringthe solubilized phase to recover the target-specific stealthnanoparticles, thereby forming a slurry of therapeutic nanoparticleshaving a diameter of about 80 nm to about 150 nm. In some embodiments,emulsifying the second phase may include emulsifying the second phase toform a coarse emulsion, and emulsifying the coarse emulsion to form afine emulsion phase. Emulsifying the second phase may be performed, forexample, by using a rotor stator homogenizer, probe sonicator, stir bar,or high pressure mixer. Emulsifying the coarse emulsion may be performedusing, for example, a high pressure homogenizer which may have multiple(e.g. 2, 3, 4 or more) interaction chambers, and with, for example, afeed pressure of about 4000 to about 8000 psi per interaction chamber.

In some embodiments, quenching may be at least partially performed at atemperature of about 5° C. or less, for example, at about 0° C. to about5° C. A quench:emulsion ratio may be about 8:1 to about 5:1, or about2:1 to about 40:1.

Exemplary drug solubilizers for use in the disclosed methods may includeTween 80, Tween 20, polyvinyl pyrrolidone, cyclodextran, sodium dodecylsulfate, or sodium cholate. In some embodiments, a drug solubilizer isselected from the group consisting of diethylnitrosamine, sodiumacetate, urea, glycerin, propylene glycol, glycofurol,poly(ethylene)glycol, bris(polyoxyethyleneglycolddodecyl ether, sodiumbenzoate, and sodium salicylate. The ratio of drug solubilizer totherapeutic agent may be about 100:1 to about 10:1.

In an embodiment, a method may include filtering the solubilized phasethat includes nanoparticles using e.g., tangential flow filtrationsystem. Filtering may be conducted, for example, at a first temperatureof about 0° C. to about 5° C., and then at a second temperature of about20° C. to about 30° C. Alternatively, filtering may be conducted, forexample, at a first temperature of about 20° C. to about 30° C., andthen at a second temperature of about 0° C. to about 5° C. In someembodiments, filtering comprises processing about 1 to about 6diavolumes at about 0° C. to about 5° C. and processing at least onediavolume at about 20° C. to about 30° C., for example, filtering mayinclude processing about 1 to about 6 diavolumes at about 0° C. to about5° C. and processing about one diavolume to about 15 diavolumes at about20° C. to about 30° C. In an embodiment, filtering may includeprocessing different diavolumes at different distinct temperatures. Thesolubilized phase may be purified before said filtering to substantiallyremove said organic solvent, unencapsulated therapeutic agent, and/ordrug solubilizer.

Disclosed methods may comprise sterile filtering of the slurry oftherapeutic nanoparticles using a filtration train at a controlled rate.For example, a filtration train may be used that comprises a depthfilter and a sterile filter.

Also provided herein is a method of preparing a plurality of therapeuticnanoparticles, comprising combining a therapeutic agent, a firstpolymer, and a second polymer, with an organic solvent to form a firstorganic phase; combining the first organic phase with a first aqueoussolution to form a second phase; emulsifying the second phase to form anemulsion phase; quenching the emulsion phase to form a quenched phase;adding a drug solubilizer to the quenched phase to form a solubilizedphase of unencapsulated therapeutic agent; and filtering the solubilizedphase using tangential flow filtration with constant volumediafiltration wherein at least one diavolume is exposed to about 25° C.after a different diavolume is exposed to about −5° C. to about 10° C.For example, filtering ma y include processing about 2 to about 5diavolumes at about 0° C. to about 5° C., and then processing at leastone diavolume at 25° C. for at least a period of time.

Provided herein are methods of forming therapeutic nanoparticles thatmay be stable for at least 2 days at 25° C. at about 10 mg/mlconcentration. Formed therapeutic nanoparticles using disclosed methodsmay release less than 10% by weight of therapeutic agent over at least 5days at 25° C. In some embodiments, a formed therapeutic nanoparticleusing a disclosed method may e.g., encapsulate about 2 to about 30percent therapeutic agent.

In some embodiments, methods of preparing a plurality of disclosedtherapeutic nanoparticles, are provided comprising combining atherapeutic agent, a first polymer (e.g., PLGA-PLA copolymer or PLA),and a second polymer (e.g. PLA, PLGA, or PEG, or copolymers thereof),and optionally a third polymer (e.g. PLA or PLGA not bound to a ligand)wherein the first polymer is bound to a ligand having a molecular weightof less than about 1000 g/mol, for example, a low-molecular weightligand e.g., a PSMA ligand. Such low-molecular weight PSMA ligand may beselected from the group consisting of compounds I, II, III and IV:

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof; wherein

m and n are each, independently, 0, 1, 2 or 3;

p is 0 or 1;

R¹, R², R⁴ and R⁵ are each, independently, selected from the groupconsisting of substituted or unsubstituted alkyl, substituted orunsubstituted aryl, and any combination thereof; and

R³ is H or CH₃;

wherein R¹, R², R⁴ or R⁵ comprise a point of covalent attachment to thenanoparticle. For example, R¹, R², R⁴ and R⁵ may be each, independently,C₁₋₆-alkyl or phenyl, or any combination of C₁₋₆-alkyl or phenyl, whichare independently substituted one or more times with OH, SH, NH₂, orCO₂H, and wherein the alkyl group may be interrupted by N(H), S or O. Inanother embodiment, for example, R¹, R², R⁴ and R⁵ are each,independently, CH₂-Ph, (CH₂)₂—SH, SH, (CH₂)₂C(H)(NH₂)CO₂H,CH₂C(H)(NH₂)CO₂H, CH(NH₂)CH₂CO₂H, (CH₂)₂C(H)(SH)CO₂H, CH₂—N(H)-Ph,O—CH₂-Ph, or O—(CH₂)₂-Ph, wherein each Ph may be independentlysubstituted one or more times with OH, NH₂, CO₂H or SH. Exemplarylow-molecular weight PSMA ligand may be selected from the groupconsisting of

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof; and wherein the NH₂, OH or SH groups serve as thepoint of covalent attachment to the first particle, or may be selectedfrom the group consisting of

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof; wherein R is independently selected from the groupconsisting of NH₂, SH, OH, CO₂H, C₁₋₆-alkyl that is substituted withNH₂, SH, OH or CO₂H, and phenyl that is substituted with NH₂, SH, OH orCO₂H,

and wherein R serves as the point of covalent attachment to the firstpolymer. Exemplary ligands include

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof; any of which may be further substituted with NH₂, SH,OH, CO₂H, C₁₋₆-alkyl that is substituted with NH₂, SH, OH or CO₂H, orphenyl that is substituted with NH₂, SH, OH or CO₂H, wherein thesefunctional groups serve as the point of covalent attachment to the firstpolymer, for example, a low-molecular weight PSMA ligand may be

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof; wherein the NH₂ groups serve as the point of covalentattachment to the first polymer.

In some embodiments, methods of preparing a plurality of disclosedtherapeutic nanoparticles, are provided comprising combining atherapeutic agent, a first polymer (e.g., PLGA-PLA copolymer or PLA),and a second polymer (e.g. PLA, PLGA, or PEG, or copolymers thereof),and optionally a third polymer (e.g. PLA or PLGA not bound to a ligand).In some embodiments, the therapeutic agent is docetaxel. In otherembodiments, the therapeutic agent is selected from the group consistingof chemotherapeutic agents such as doxorubicin (adriamycin),mitoxantrone, gemcitabine (gemzar), daunorubicin, procarbazine,mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU),vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel(taxotere), aldesleukin, asparaginase, busulfan, carboplatin,cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethylcamptothecin (SN38),dacarbazine, S-I capecitabine, ftorafur, 5′ deoxyfluorouridine, UFT,eniluracil, deoxycytidine, 5-azacytosine, 5-azadeoxycytosine,allopurinol, 2-chloroadenosine, trimetrexate, aminopterin,methylene-10-deazaminopterin (MDAM), oxaplatin, picoplatin, tetraplatin,satraplatin, platinum-DACH, ormaplatin, CI-973, JM-216, and analogsthereof, epirubicin, etoposide phosphate, 9-aminocamptothecin,10,11-methylenedioxycamptothecin, karenitecin, 9-nitrocamptothecin, TAS103, vindesine, L-phenylalanine mustard, ifosphamidemefosphamide,perfosfamide, trophosphamide carmustine, semustine, epothilones A-E,tomudex, 6-mercaptopurine, 6-thioguanine, amsacrine, etoposidephosphate, karenitecin, acyclovir, valacyclovir, ganciclovir,amantadine, rimantadine, lamivudine, zidovudine, bevacizumab,trastuzumab, rituximab, and 5-Fluorouracil, methotrexate, budesonide,sirolimus vincristine, and combinations thereof, or the therapeuticagent may be an siRNA.

Also provided herein are methods of treating prostate cancer in asubject in need thereof, comprising administering to the subject aneffective amount of the nanoparticle prepared by the disclosed methods.

In an embodiment, also provided herein is a therapeutic nanoparticleprepared by: emulsification of a first organic phase comprising a firstpolymer and a therapeutic agent and second phase forming an emulsionphase; wherein the emulsion phase is then quenched at a temperature ofabout 0° C. to about 5° C. forming a quenched phase; and filtration ofthe quenched phase at a first temperature of about −5° C. to about 10°C.; and filtering the quenched phase at a second temperature of about25° C.; thereby forming therapeutic nanoparticles that are stable for atleast 5 days at 25° C.

A method of stabilizing therapeutic nanoparticles is also provided in anembodiment, having a therapeutic agent comprising: providing a slurrycomprising a therapeutic agent encapsulated by nanoparticles and a drugsolubilizer; filtering the slurry at a first temperature of about −5° C.to about 10° C.; filtering the slurry at a second temperature of about25° C.

The invention claimed is:
 1. A method of preparing a plurality oftherapeutic nanoparticles, comprising: combining a therapeutic agent, afirst polymer, and optionally a second polymer, with an organic solventto form a first organic phase having about 5 to about 50% solids;combining the first organic phase with a first aqueous solution to forma second phase; emulsifying the second phase to form an emulsion phase;quenching the emulsion phase to form a quenched phase; adding a drugsolubilizer to the quenched phase to form a solubilized phase ofunencapsulated therapeutic agent; and sterile filtering the solubilizedphase to recover the therapeutic nanoparticles, thereby forming a slurryof therapeutic nanoparticles having a diameter of about 80 nm to about150 nm.
 2. The method of claim 1, wherein emulsifying the second phasecomprises: emulsifying the second phase to form a coarse emulsion, andemulsifying the coarse emulsion to form a fine emulsion phase.
 3. Themethod of claim 1, wherein the organic solvent comprises a solventchosen from: ethyl acetate, benzyl alcohol, methylene chloride,chloroform, toluene, methyl ethyl ketone, dimethyl formamide, dimethylsulfoxide, acetone, acetonitril, acetic acid, Tween 80 and Span 80, andcombinations of two or more thereof.
 4. The method of claim 1, whereinthe first aqueous solution comprises a reagent chosen from: sodiumcholate, ethyl acetate, benzyl alcohol or combinations thereof.
 5. Themethod of claim 2, wherein emulsifying the second phase comprises usinga rotor stator homogenizer, probe sonicator, stir bar, or high pressurehomogenizer.
 6. The method of claim 2, wherein emulsifying the coarseemulsion comprises using a high pressure homogenizer.
 7. The method ofclaim 6, wherein emulsifying the coarse emulsion comprises about 2 toabout 3 passes through the homogenizer.
 8. The method of claim 6,wherein the homogenizer feed pressure is about 2000 to about 8000 psiper interaction chamber.
 9. The method of claim 6, wherein thehomogenizer comprises multiple interaction chambers.
 10. The method ofclaim 1, wherein quenching is performed at about 0° C. to about 5° C.11. The method of claim 1, wherein the quench: emulsion ratio is about8:1 to about 5:1.
 12. The method of claim 1, wherein the quench:emulsion ratio is about 2:1 to about 40:1.
 13. The method of claim 1,wherein the drug solubilizer is selected from the group consisting ofTween 80, Tween 20, polyvinyl pyrrolidone, cyclodextran, sodium dodecylsulfate, and sodium cholate.
 14. The method of claim 1, wherein the drugsolubilizer is selected from the group consisting of diethylnitrosamine,sodium acetate, urea, glycerin, propylene glycol, glycofurol,poly(ethylene)glycol, bris(polyoxyethyleneglycolddodecyl ether, sodiumbenzoate, and sodium salicylate.
 15. The method of claim 1, wherein theratio of drug solubilizer to therapeutic agent is about 100:1 to about10:1.
 16. The method of claim 1, wherein filtering comprises using atangential flow filtration system.
 17. The method of claim 1, whereinfiltering comprises filtering at a first temperature of about 0° C. toabout 5° C.
 18. The method of claim 17, further comprising filtering ata second temperature of about 20° C. to about 30° C.
 19. The method ofclaim 18, wherein filtering comprises processing about 1 to about 6diavolumes at about 0° C. to about 5° C. and processing at least onediavolume at about 20° C. to about 30° C.
 20. The method of claim 18,wherein filtering comprises processing about 1 to about 6 diavolumes atabout 0° C. to about 5° C. and processing about one diavolume to about15 diavolumes at about 20° C. to about 30° C.
 21. The method of claim 1,further comprising purifying the solubilized phase before said filteringto remove said organic solvent, unencapsulated therapeutic agent, and/ordrug solubilizer.
 22. The method of claim 1, further comprisingsterilely filtering the slurry of therapeutic nanoparticles using afiltration train at a controlled rate.
 23. The method of claim 22,wherein the filtration train comprises a depth filter and a sterilefilter.
 24. The method of claim 1, wherein the first polymer is aPLGA-PLA or PLA, and the second polymer is a PLGA-PLA-PEG copolymer, aPLGA-block-PEG copolymer, or a PLA-block-PEG copolymer.
 25. The methodof claim 1, wherein the therapeutic agent is docetaxel.